CypExpress 3A4 Catalyzed Conversion of Testosterone (TE) to 6β- Hydroxytestosterone (HT)

Size: px
Start display at page:

Download "CypExpress 3A4 Catalyzed Conversion of Testosterone (TE) to 6β- Hydroxytestosterone (HT)"

Transcription

1 TM CASE STUDY CypExpress 3A4 Catalyzed Conversion of to Shuvendu Das, 1 Enrique Martez, 2 and Mani Subramanian 1 1 Center for Biocatalysis and Bioprocessg, University of Iowa 2 Oxford Biomedical Research, Inc. 215 Oxford Biomedical Research, Inc.

2 Introduction: These studies were performed usg the FDA- recommended substrate Testosterone to evaluate the utility of CypExpress3A4 for drug metabolism and disposition studies. Introduction: These studies were performed usg the FDA- recommended substrate Introduction: Testosterone These studies to evaluate were performed the utility usg of the CypExpress3A4 FDA- recommended for drug metabolism substrate Dextromethorphan and disposition to studies. evaluate the utility of CypExpress2D6 for drug metabolism and disposition studies. Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, Reaction volume 2 μl, Reaction time 2 to 4 h Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, Metabolite Identification 5 mm KPi 24- well buffer microplates: ph 7.5, 3 C, 5 6 1, rpm μm DOM, microplate 2-1 shaker, Reaction mg/ml CypExpress2D6, volume 2 μl, 5 Reaction mm KPi buffer time ph 2 7.5, to 4 3 C, h 2-6 rpm microplate shaker, Reaction volume 2 μl, Reaction time 3 to 4 h or as specified. Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm glass glass tube tube usg the the followg followg fal fal concentrations: concentrations: Substrate = 5 2 to 1, 5 μm TE DOM. (5 µm for scale- up). Unless otherwise CypExpress 2D6 specified, = 1 mg/ml TE was added to the reaction mixture as a concentrated solution DMSO). Buffer = 5 mm KPi ph 7.5 CypExpress NADP+, G6P = 3A4 NONE = 1 ADDED mg/ml for 1 st cycle; see details for additional cycles Buffer G6PDH = and 5 Mg++ 1 = mm NONE KPi, ADDED. or Tris- HCl Contaed (as specified CypExpress tables), system. ph 7.5 The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, G6PDH and agitated and Mg++ by rotation = NONE at 225 ADDED. rpm for Contaed 3. h. CypExpress system. Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm glass tube usg the followg fal concentrations: Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a concentrated solution DMSO). CypExpress 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, A. The and entire agitated suspension by rotation was extracted at 225 rpm and subjected for 3. h. to HPLC (figure 1). Or At the end of the reaction period, either B. CypExpress2D6 was pelleted by centrifugation at 14, x g. A. The a. entire The supernatant suspension from was the extracted first cycle and was subjected analyzed by to HPLC. HPLC (figure 1). Or which the entire suspension was extracted and subjected to HPLC. B. CypExpress3A4 was pelleted by centrifugation at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 2 HPLC. A. The entire suspension was extracted and subjected to HPLC (figure 1). Or B. CypExpress3A4 was pelleted by centrifugation at 14, x g. which the entire suspension was extracted and subjected to HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 2

3 CypExpress 3A4 Conversion of Testosterone to 6β- Hydroxytestosterone Conversion of TE to HT a sgle reaction cycle: CypExpress 3A4 Introduction: (1 mg/ml) These catalyzed studies conversion were performed of 2 usg μm the FDA- recommended to substrate 6β- Hydroxytestosterone Stability Testosterone of CypExpress2D6 to (HT) evaluate stored a sgle at the - 8 C: utility 3 hr. CypExpress reaction of CypExpress3A4 cycle is a novel 5 vitro for μm drug TE, P45 5 mm metabolism KPi catalytic of ph 7.5 and system at disposition 3 C with was multiple vestigated studies. components by (recombant RP- HPLC after human extraction 2D6, of the entire reaction recombant mixture human with HPLC NADP Mobile oxidoreductase, Phase contag cofactors, 79% G6PDH, (v/v) and methanol, antioxidants 1% (v/v) acetic an acid, eukaryotic and 2% matrix). (v/v) Hence, water it was 1:1 important (v/v) ratio. to The fully results characterize are shown various figure 1 which aspects the of the black system le and represents its activity. the The HT stability standard, upon the storage light under blue various le represents results conditions obtaed was usg evaluated. a vector Not unsurprisgly, control (no 3A4), it proved and the very red stable le when represents stored results obtaed as a dry usg powder CypExpress3A4. at - 8 C (figure 1): 1 SPD-M1Avp-24 nm 1uM 6-betaOHTest stand 1uLd Isolation RM 1uL GO Cont 1uL 1 6β- Hydroxytestosterone 8 (HT) 7 7 Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml 6 6 CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, 5 5 Reaction volume 2 μl, Reaction time 2 to 4 h TE mau mau Pilot- scale reactions were performed a total volume of 1. ml a 2x mm HT glass tube usg the followg fal concentrations: 2 Flow- Thru Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise 1 1 Although not recommended, specified, we TE have was also added observed to the that reaction dry CypExpress mixture powder as a can be stored at room concentrated temperature solution for up to 3 weeks, DMSO). and that CypExpress suspensions buffer can be frozen and thawed Mutes up to four times without demonstrable CypExpress loss 3A4 of activity. = 1 mg/ml Mutes Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 Dose- dependent conversion of DOM to DOH by CypExpress2D6 24 well Yield of HT from TE a sgle reaction NADP+, G6P = NONE ADDED for 1 st cycle: Usg the conditions stated microplates: Investigations to P45- mediated cycle; metabolism see details of new chemical for additional cycles entities, above cludg (5 µm determation TE, 1 mg/ml of the CypExpress3A4, metabolism (if any) 3 C, catalyzed 3 hr., 5 by mm specific Pi ph 7.5), P45 the isoforms G6PDH yield of and HT the Mg++ a identification sgle = NONE reaction ADDED. of the cycle metabolite(s) Contaed was: produced CypExpress is typically system. performed microplates followed by HPLC- MS analysis. The suitability of 87.9 µm = 17.6% conversion The CypExpress 2 x 15 for mm such reaction applications tube was vestigated placed usg a tilted CypExpress2D6 position on (1 a shaker platform at mg/ml) 3 C, and conversion agitated of by DOM rotation to DOH at mm rpm phosphate for 3. buffer h. ph 7.5. The production Comparison of DOH of from the 5 catalytic µm DOM efficiency by different and concentrations product purify of for pre- washed At CypExpress2D6 the vs. end unwashed of the at reaction varyg CypExpress3A4: cubation period, either times All conditions (3 C) was were examed identical (figure to those 2). the first Although the reaction rate slows with time, the greatest product yield was obtaed set of experiments, except that a parallel experiment the CypExpress3A4 was usg A. 1 The mg/ml entire of suspension CypExpress2D6 was for extracted 3-4 hrs. This and is subjected a convenient to HPLC time for (figure high- 1). pre- washed by (a) suspendg reaction buffer and allowed to stand for throughput microplate studies. various Or periods of time (see graph and chart) followed by (b) centrifugation at 14, x g, (c) removg the supernatant, and (d) resuspendg the B. CypExpress3A4 fresh was reaction pelleted buffer(g6p)? by centrifugation. Results at 14, comparg x g. the HPLC profiles a. and The the supernatant catalytic efficiency from the are first presented cycle was Figure analyzed 2 and by HPLC. Table Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 3 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page mau

4 CypExpress 3A4 Conversion of Testosterone to 6β- Hydroxytestosterone Figure 2. HPLC analysis of TE - > HT catalyzed by unwashed CypExpress3A4 (light Introduction: blue) and CypExpress3A4 These studies followg were performed washg usg 5 (dark the red), FDA- recommended 1 (dark blue), 15 substrate (dark green) Testosterone or 2 m (light to evaluate green). the utility of CypExpress3A4 for drug SPD-M1Avp-24 nm metabolism and disposition studies. h wash 1uL New 1 MP 1uL 5m wash 1uL 1m wash 1uL 15m wash 1uL 2m wash 1uL 6 h wash 1uL PM.dat new 1 mp 1ul pm.dat 5m wash 1ul pm.dat 1m wash 1ul pm.dat 15m wash 1ul pm.dat 2m wash 1ul pm.dat mau Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml 15 Determation 15 of Km and Vmax of CypExpress2D6 for DOM: Km and Vmax values CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, were obtaed 1 for CypExpress2D6 (1 mg/ml) cubated for 4 hrs at 3 C with 1 Reaction with DOM volume rangg 2 from μl, 5 Reaction 1, µm. A time Leweaver- Burke 2 to 4 h plot of the results thus 5 5 obtaed is presented figure 3, and gave a Km of 34 µm and a Vmax of 714 Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm µm/hr/gram of CypExpress. However, as shown Figure 2 and Table 1 (below), at glass 1 tube mg/ml 1.75 usg CypExpress 2. the 2.25 followg 2.5 2D6, the fal 3.25 reaction 3.5 concentrations: 3.75 slows 4. significantly after hr Mutes Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a concentrated solution DMSO). CypExpress 3A4 wash μm of TE Reaction hrs μm of HT* CypExpress 3A4 = 1 mg/ml Buffer = 5 None 1 mm KPi, or 5 Tris- HCl (as specified 3 tables), ph m G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. 1 m The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. 15 m m A. The entire suspension was extracted and subjected to HPLC (figure 1). CONCLUSION: Or Pre- washg CypExpress reduces the size of the flow- thru peak comprised of very hydrophilic components, but does not significantly alter the B. catalytic CypExpress3A4 efficiency of was CypExpress. pelleted by centrifugation at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 4 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page mau

5 Applications of CypExpress for P45 profilg & metabolite identification: Introduction: Investigations These to P45- mediated studies were metabolism performed of usg new the chemical FDA- recommended entities, substrate cludg The data Testosterone presented determation Table to of evaluate 1 the also metabolism demonstrate the utility (if the any) of reproducibility CypExpress3A4 catalyzed by of specific these for drug P45 metabolism isoforms microplate and studies. and the disposition identification studies. of the metabolite(s) produced is typically performed microplates followed by HPLC- MS analysis. The suitability of CypExpress for such applications was vestigated usg CypExpress3A4 (1 mg/ml) conversion CypExpress of 2D6 TE to HT μm of 5 DOM mm phosphate Reaction hrs buffer ph μm 7.5. of DOH The conversion of various concentrations of TE to HT a 2 µl reaction volume 24 well microplates 1 mg/ml cubated with 25 shakg for 3 2 hr at 3 C is 54.6 shown ± 1.7 Table 2: Sample 1 mg/ml TE (μm) 5 Reaction 2 Time (h) 67.1 ± 2.2 HT (μm) Testosterone 1 (TE) mg/ml ± mg/ml ± 1. Metabolite 3 Identification well microplates: μm, mg/ml CypExpress3A4, 4 5 mm 25 KPi buffer ph 7.5, 3 C, 3. 6 rpm microplate 83.3 shaker, CONCLUSION: CypExpress is well suited for microplate reactions. NOTE: for Reaction longer volume 2 μl, Reaction time 2 to 4 h 5 reaction times 5 it is important to restrict evaporation, 3. e.g. by performg a humidified environment. Pilot- scale 6 reactions were 1 performed a total 3. volume of 1. ml a 2x15 mm glass Scale- up tube usg conversion the followg of DOM fal to DOH concentrations: a sgle reaction cycle: The rate of CONCLUSION: conversion 5 Substrate CypExpress μm DOM to DOH = 2 to 5 is well by μm TE suited CypExpress (5 for µm microplate 2D6(1 for scale- up). reactions. mg/ml) at Unless NOTE: 3 C otherwise for a of a longer 2 ml reaction reaction times volume specified, it was is important vestigated TE was to restrict by RP- HPLC added to evaporation, after extraction the reaction e.g. mixture by of performg entire as a reaction mixture with HPLC Mobile Phase contag 79% (v/v) methanol, 1% (v/v) a humidified environment. acetic acid, and 2% (v/v) concentrated water 1:1 solution (v/v) ratio. DMSO). The results are shown figure Stability 4: CypExpress of CypExpress3A4 3A4 = 1 stored mg/ml at - 8 C: CypExpress is a novel vitro P45 catalytic system with multiple components (recombant human 3A4, recombant Buffer = human 5 1 NADP mm oxidoreductase, KPi, or Tris- HCl cofactors, (as specified G6PDH, and tables), antioxidants ph an eukaryotic matrix). Hence, it was important NADP+, G6P = NONE ADDED for 1 st to fully characterize various cycle; see details for additional cycles aspects of the system 12 and its activity. The stability upon storage under various conditions G6PDH was and evaluated. Mg++ = Not NONE unsurprisgly, ADDED. Contaed it proved very CypExpress stable when system. stored 1 as a dry powder at - 8 C (figure 3): The 2 x 15 mm 8 reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. 6 At the end of the reaction 4 period, either Dextrorphan (µm) A. The entire 2 suspension was extracted and subjected to HPLC (figure 1). Or B. CypExpress3A4 was pelleted Time by centrifugation (hr) at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 5 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 5

6 Although not recommended, we have also observed that dry CypExpress powder Introduction: can be stored at These room studies temperature were performed up to 3 weeks, usg and the that FDA- recommended CypExpress substrate The suspensions HPLC Testosterone profiles buffer of aliquots can to be evaluate of frozen the reaction the and utility thawed mixture of up CypExpress3A4 taken to four at times these without time for tervals drug metabolism are demonstrable presented and loss Figure disposition of activity. 5: studies. Effects of solvents: Testosterone and many pharmaceutically active substances are poorly soluble aqueous solutions, so they are typically added to vitro P45 reaction systems as a concentrated solution a water- miscible solvent. The general guidance has been that the fal concentration of that solvent the to vitro P45 reaction system should be kept below 1%. For CypExpress, we vestigated the effect of the solvent used to dissolve TE on the conversion of TE to HT. The results obtaed are shown figure 4: 14 Metabolite 12 Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, Reaction 1.4% volume 2 μl, Reaction time 2 to 4 h.8% Pilot- scale 8 reactions were 1.7% performed a total volume of 1. ml a 2x15 mm glass tube usg the followg 2.5% fal concentrations: Figure Substrate 6 zooms 6 = on 2 the to peak 5 obtaed μm TE (5 for DOH µm production for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a 4 concentrated solution DMSO). 6β-Hydroxytestosterone (μm) CypExpress 2 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 NADP+, G6P = Methanol NONE ADDED for 1 st DMF cycle; see details for DMSO additional cycles CONCLUSIONS: G6PDH and As Mg++ has been = NONE reported ADDED. for other Contaed vitro P45 CypExpress catalytic systems, system. the use of methanol as a solvent should be avoided. However, at least for this The 3A4 2 system x 15 with mm reaction TE as substrate, tube was CypExpress placed is a activity tilted position is mimally on a hibited shaker platform by at concentrations 3 C, and agitated of DMSO by rotation and DMF at up 225 to at rpm least for 2.5%. 3. h. At Effect the end of buffer of the reaction the production period, either of HT from TE: The production of HT Tris and Phosphate buffers was compared (Table 3): A. The entire suspension was extracted and subjected to HPLC (figure 1). HT Buffer Or TE (μm) Reaction Time (h) (μm) KPi, B. 1 CypExpress3A4 mm was pelleted 5 by centrifugation 3. at 14, x g Tris- HCl, b. 1 The mm pellet was resuspended 5 fresh 3. buffer contag G6P, but no 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 6 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 6

7 CypExpress CypExpress 3A4 3A4 Conversion Conversion of Testosterone of Testosterone to 6β- Hydroxytestosterone to 6β- Hydroxytestosterone CONCLUSION: Although high levels of conversion of TE to HT were obtaed Introduction: reactions catalyzed These by studies CypExpress3A4 were performed both buffers, usg the for this FDA- recommended isoform and this Figure substrate 7 zooms Testosterone the conversion the time- dependent to Tris- HCl evaluate buffer the dimution utility is more of efficient of CypExpress3A4 the peak than obtaed the same for for drug reaction the metabolism substrate performed DOM and KPi disposition studies. Increased yields for the production of HT from TE by CypExpress3A4 multiple reaction cycles: Rationale: Studies of the stability and activity of CypExpress systems expressg various recombant human P45 enzymes have shown that: Significant CypExpress activity is retaed after a sgle reaction cycle CypExpress typically adsorbs large quantities of relatively hydrophobic Testosterone substrates from (TE) the reaction mixture, but releases most of the more hydrophobic product(s) to the buffer. Reaction Low speed Conditions: centrifugation after one reaction cycle pellets CypExpress, which Metabolite contas Identification significant quantities 24- well of unreacted microplates: substrate 25-5 and μm, some 2-1 product. mg/ml CypExpress3A4, Typically, the 5 total mm product KPi buffer yield ph is greater 7.5, 3 C, for 6 multi- cycle rpm reactions microplate vs. shaker, Reaction longer volume cubations 2 μl, for Reaction a sgle cycle. time 2 to 4 h Additional reaction cycles convert substantial amounts of the retaed Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm substrate to product thereby creasg overall metabolite yield. glass tube usg the followg fal concentrations: Sgle cycle scale- up synthesis of TE: A 2 ml reaction was performed usg a Yield TE concentration of Substrate DOH from = DOM of 2 25 to 5 µm a sgle and μm reaction withdrawg TE (5 cycle: µm aliquots Usg for scale- up). the at conditions different Unless stated time otherwise tervals above (5 to follow µm DOM, the time 1 mg/ml course specified, of CypExpress2D6, the TE reaction. was added HPLC 3 C, 3 analysis to hr., the 5 reaction mm of the Pi ph production mixture 7.5), the yield as of a HT of from 5.65 TE DOM (figure a sgle 5) dicates concentrated reaction that cycle the (extracted solution reaction reaction rate DMSO). slows suspension). appreciably If the after ~ 2hr. CypExpress is centrifuged, the quantity of DOH recoverable from the supernatant fraction CypExpress was 3. mg. The 3A4 = 1 mg/ml Increased Buffer yield = 5 for the 1 production mm KPi, or of Tris- HCl DOH from (as DOM specified by CypExpress2D6 tables), ph 7.5 multiple reaction cycles: Rationale: Studies of the stability and activity of CypExpress systems expressg various G6PDH recombant and Mg++ human = NONE P45 ADDED. enzymes Contaed have shown that: CypExpress system. Significant CypExpress activity is retaed after a sgle reaction cycle The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform CypExpress typically adsorbs large quantities of relatively hydrophobic at 3 C, substrates and agitated from by the rotation reaction at mixture, 225 rpm but releases for 3. most h. of the more hydrophobic product(s) to the buffer. yield Low speed centrifugation after one reaction cycle pellets CypExpress, which of A. HT The contas entire significant suspension quantities was extracted of unreacted and substrate subjected and some to HPLC product. (figure 1). Typically, the total product yield is greater for multi- cycle reactions vs. Or longer cubations for a sgle cycle. Additional reaction cycles convert substantial amounts of the retaed B. CypExpress3A4 substrate to product was thereby pelleted creasg by centrifugation overall metabolite at 14, yield. x g. obtaed a. at The supernatant from the first cycle was analyzed by HPLC. different b. time The tervals pellet this was experiment resuspended is shown fresh Figure buffer 6 contag below. G6P, but no 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 7 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 7

8 CypExpress CypExpress 3A4 3A4 Conversion Conversion of Testosterone of Testosterone to 6β- Hydroxytestosterone to 6β- Hydroxytestosterone Introduction: These 8 studies were performed usg the FDA- recommended substrate Testosterone 7 to evaluate the utility of CypExpress3A4 for drug metabolism and disposition 6 studies Time (hr) Metabolite Scale- up production Identification of TE 24- well a second microplates: catalytic cycle: 25-5 A 2 ml μm, reaction 2-1 was mg/ml performed usg a TE concentration of 25 µm. After a 3 hr. first reaction cycle, CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, the CypExpress was pelleted, and the supernatant harvested. The pelleted Reaction volume 2 μl, Reaction time 2 to 4 h CypExpress was stored overnight at 4C, then resuspended fresh buffer Pilot- scale contag reactions G6P but no were additional performed NADP+ a or total TE substrate. volume of This 1. suspension ml a 2x15 was mm cubated at 3 C and aliquots withdrawn at different time tervals to follow glass tube usg the followg fal concentrations: the time course of the reaction by HPLC. The results obtaed (figure 6) support the production Substrate of = substantial 2 to 5 μm additional TE (5 TE µm for for at least scale- up). an additional Unless 2.5 otherwise hr. specified, TE was added to the reaction mixture as a concentrated solution DMSO). 6β-Hydroxytestosterone (µm) CypExpress 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. A. The entire suspension was extracted and subjected to HPLC (figure 1). Or Discussion: CypExpress3A4 retas activity after several hours and the yields of metabolite B. CypExpress3A4 scale- up can was be pelleted significantly by centrifugation creased by multiple at 14, reaction x g. cycles. which the entire suspension was extracted and subjected to HPLC. 215 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered the US and other countries. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 8

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)

More information

Announcements Columbus Day 10/8 No class, 10/9 BU Monday!

Announcements Columbus Day 10/8 No class, 10/9 BU Monday! Announcements Columbus Day 10/8 No class, 10/9 BU Monday! Make up section for Tuesday discussion: Wednesday 10/10, 5-6 pm, SAR 102 Monday Section: Tuesday 10/9, 10-11 am KCB 106 Wednesday Section: Wed.

More information

HighPure Maxi Plasmid Kit

HighPure Maxi Plasmid Kit HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer

More information

Chromatin Immunoprecipitation

Chromatin Immunoprecipitation Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin

More information

Peptide Antibody Production

Peptide Antibody Production Peptide Antibody Production A) Peptide BioSynthesis (http://www.biosyn.com, 800-227-0627) B) Conjugation of peptide to KLH (Imject Maleimide Activated KLH, PIERCE=Thermo #77605, 10 mg) C) Peptide affinity

More information

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep Guide to Reverse Phase SpinColumns Chromatography for Sample Prep www.harvardapparatus.com Contents Introduction...2-3 Modes of Separation...4-6 Spin Column Efficiency...7-8 Fast Protein Analysis...9 Specifications...10

More information

6 Characterization of Casein and Bovine Serum Albumin

6 Characterization of Casein and Bovine Serum Albumin 6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function

More information

Human serum albumin (HSA) nanoparticles stabilized with. intermolecular disulfide bonds. Supporting Information

Human serum albumin (HSA) nanoparticles stabilized with. intermolecular disulfide bonds. Supporting Information Human serum albumin (HSA) nanoparticles stabilized with intermolecular disulfide bonds Wentan Wang, Yanbin Huang*, Shufang Zhao, Ting Shao and Yi Cheng* Department of Chemical Engineering, Tsinghua University,

More information

Collagen analysis in fish tissue using the total collagen assay. Application note April 2013

Collagen analysis in fish tissue using the total collagen assay. Application note April 2013 Collagen analysis in fish tissue using the total collagen assay Application note April 2013 Summary The QuickZyme Total Collagen assay provides an easy and fast method to determine collagen quantities

More information

HiPer Ion Exchange Chromatography Teaching Kit

HiPer Ion Exchange Chromatography Teaching Kit HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for

More information

TransformAid Bacterial Transformation Kit

TransformAid Bacterial Transformation Kit Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection

More information

Innovative vs Traditional

Innovative vs Traditional And in the red corner, introducing the challenger NASDAQ April 4 2011 243.47 $ 1.38 $ 0.58 % 0.07 $ 0.02 $ 39.4 % Technology DVD Digital Streamline Instant Play Internet Access Unlimited Selection No Late

More information

For the rapid, sensitive and accurate measurement of Glutamate Dehydrogenase activity in various samples

For the rapid, sensitive and accurate measurement of Glutamate Dehydrogenase activity in various samples ab102527 Glutamate Dehydrogenase Detection Kit Instructions for Use For the rapid, sensitive and accurate measurement of Glutamate Dehydrogenase activity in various samples This product is for research

More information

Single cell cloning of suspension cells with Calcein-AM and the Cellavista System

Single cell cloning of suspension cells with Calcein-AM and the Cellavista System Single cell cloning of suspension cells with Calcein-AM and the Cellavista System Introduction Single cell cloning (SCC) represents a critical step in cell line development for the production of biopharmaceuticals.

More information

Chromatin Immunoprecipitation (ChIP)

Chromatin Immunoprecipitation (ChIP) Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without

More information

TECHNICAL BULLETIN. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets. Catalog Number T9424 Store at room temperature.

TECHNICAL BULLETIN. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets. Catalog Number T9424 Store at room temperature. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets Catalog Number T9424 Store at room temperature. TECHNICAL BULLETIN Product Description TRI Reagent is a quick and convenient

More information

Human Pancreatic Beta Cell Lines Culture Protocols (provided by the depositor)

Human Pancreatic Beta Cell Lines Culture Protocols (provided by the depositor) Human Pancreatic Beta Cell Lines Culture Protocols (provided by the depositor) 1. Thawing Human Cell Lines for culture: Locate vials in liquid nitrogen and quickly remove with forceps. Take vial to sterile

More information

lung cancer targeted photodynamic therapy and imaging

lung cancer targeted photodynamic therapy and imaging 99m Tc-Hematoporphyrin linked albumin nanoparticles for lung cancer targeted photodynamic therapy and imaging Su-Geun Yang, Ji-Eun Chang, Byungchul Shin, Sanghyun Park, Kun Na and Chang-Koo Shim* *Corresponding

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

An In-Gel Digestion Protocol

An In-Gel Digestion Protocol An In-Gel Digestion Protocol This protocol describes the digestion of a protein present in an SDS-PAGE gel band with trypsin. The band can be taken from either a 1D or 2D electrophoresis gel. Reagents

More information

GLUCOSE OXIDASE

GLUCOSE OXIDASE www.megazyme.com GLUCOSE OXIDASE ASSAY PROCEDURE K-GLOX 04/14 (200 Assays per Kit) or (1960 Auto-Analyser Assays per Kit) or (2000 Microplate Assays per Kit) Megazyme International Ireland 2014 INTRODUCTION:

More information

Enzymes: Amylase Activity in Starch-degrading Soil Isolates

Enzymes: Amylase Activity in Starch-degrading Soil Isolates Enzymes: Amylase Activity in Starch-degrading Soil Isolates Introduction This week you will continue our theme of industrial microbiologist by characterizing the enzyme activity we selected for (starch

More information

Human Free Testosterone(F-TESTO) ELISA Kit

Human Free Testosterone(F-TESTO) ELISA Kit Human Free Testosterone(F-TESTO) ELISA Kit Catalog Number. MBS700040 For the quantitative determination of human free testosterone(f-testo) concentrations in serum, plasma. This package insert must be

More information

BUFFERS and MEDIAS Coomassie Blue Staining Solution Coomassie blue Destaining Solution DMEM Normal Cell Culture Media

BUFFERS and MEDIAS Coomassie Blue Staining Solution Coomassie blue Destaining Solution DMEM Normal Cell Culture Media BUFFERS and MEDIAS Coomassie Blue Staining Solution 2 g Coomassie Blue 2 L Methanol or Ethanol * 1.6 L 400 ml Glacial acetic acid *If you will be microwaving the gel in staining solution for rapid staining

More information

NimbleGen DNA Methylation Microarrays and Services

NimbleGen DNA Methylation Microarrays and Services NimbleGen DNA Methylation Microarrays and Services Sample Preparation Instructions Outline This protocol describes the process for preparing samples for NimbleGen DNA Methylation microarrays using the

More information

Overview Purpose & Scope

Overview Purpose & Scope Page 1 of 5 Overview Purpose & Scope This procedure describes a method to extract high quality genomic DNA from processed plant tissues (e.g., leaf, grain, or seed). Monsanto has optimized and performed

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

Plant Genomic DNA Extraction using CTAB

Plant Genomic DNA Extraction using CTAB Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however

More information

ab83369 Alkaline Phosphatase Assay kit (Colorimetric)

ab83369 Alkaline Phosphatase Assay kit (Colorimetric) ab83369 Alkaline Phosphatase Assay kit (Colorimetric) Instructions for use: For the rapid, sensitive and accurate measurement of Alkaline Phosphatase in various samples. This product is for research use

More information

SDS-PAGE. (June 23, 2005)

SDS-PAGE. (June 23, 2005) SDS-PAGE (June 23, 2005) GATHER REAGENTS & MATERIALS 30% acrylamide/0.8% bisacrylamide (30:1) 4X Tris. Cl/SDS, ph 8.8 4X Tris. Cl/SDS, ph 6.8 Ammonium persulfate, 10% (Make fresh each time.) SDS electrophoresis

More information

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control is a is a state of the art transfection reagent, specifically designed for the transfer of sirna and mirna into a variety of eukaryotic cell types. is a state of the art transfection reagent, specifically

More information

General western blot protocol. Guidance for running an efficient and accurate experiment

General western blot protocol. Guidance for running an efficient and accurate experiment blot protocol Guidance for running an efficient and accurate experiment Contents Introduction Solution and reagents Sample lysis Sample preparation Loading and running the gel Antibody staining Useful

More information

SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual

SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Toll Free: 866-252-7771 752A Lincoln Blvd. Phone: 732-469-7771 Fax: 732-469-7782 Middlesex, NJ 08846 Web: www.purebiotechllc.com SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Product: SOLIDscript

More information

IN VITRO BINDING BIOEQUIVALENCE STUDY SUMMARY TABLES AND SAS TRANSPORT FORMATTED TABLES FOR DATASET SUBMISSION

IN VITRO BINDING BIOEQUIVALENCE STUDY SUMMARY TABLES AND SAS TRANSPORT FORMATTED TABLES FOR DATASET SUBMISSION IN VITRO BINDING BIOEQUIVALENCE STUDY SUMMARY TABLES AND SAS TRANSPORT FORMATTED TABLES FOR DATASET SUBMISSION I. For Calcium Acetate Drug Products Table I.1 Submission Summary * Drug Product Name Strength(s)

More information

Affinity Chromatography

Affinity Chromatography PR100-02 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Affinity Chromatography Teacher s Guidebook (Cat. # BE-417) think proteins! think

More information

Oasis HLB Cartridges and 96-Well Plates

Oasis HLB Cartridges and 96-Well Plates CONTENTS I. INTRODUCTION II. SAMPLE PRE-TREATMENT a. Biological Samples b. Solid Samples: Soil, Whole Foods, Tissue c. Aqueous Samples: Water, Beverages d. Non-Aqueous Liquid III. SOLID PHASE EXTRACTION

More information

Optimizing Performance of the Transcreener ADP Assay for the BioTek Synergy 2 and 4 Multi-Mode Microplate Readers

Optimizing Performance of the Transcreener ADP Assay for the BioTek Synergy 2 and 4 Multi-Mode Microplate Readers Optimizing Performance of the Transcreener ADP Assay for the BioTek Synergy 2 and 4 Multi-Mode Microplate Readers Brad Larson 1, Karen Kleman-Leyer 1, Xavier Amouretti 2 1 BellBrook Labs, Madison, WI,

More information

Amaxa 4D-Nucleofector Protocol for Mouse Embryonic Stem [ES] Cells For 4D-Nucleofector X Unit Transfection in suspension

Amaxa 4D-Nucleofector Protocol for Mouse Embryonic Stem [ES] Cells For 4D-Nucleofector X Unit Transfection in suspension For 4D-Nucleofector X Unit Transfection in suspension Cells derived from mouse blastocysts; round cells, growing in clumps s 1. This protocol is meant to provide an outline for the handling and the Nucleofection

More information

RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method)

RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method) Immune Tolerance Network RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method) Author: Paul Wallace, Director, RPCI Laboratory of Flow Cytometry Approved by: Paul

More information

Determination of Anabolic Steroids in Horse Urine by SPE and LC-MS/MS

Determination of Anabolic Steroids in Horse Urine by SPE and LC-MS/MS Summary: Determination of Anabolic Steroids in Horse Urine by SPE and LC-MS/MS UCT Part Numbers: CUNAX226 - Clean-Up C8+NAX, 2mg/6mL BETA-GLUC- ml Beta-Glucuronidase Enzyme, liquid form SLAQ1ID21-3UM -

More information

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)

More information

TIANquick Mini Purification Kit

TIANquick Mini Purification Kit TIANquick Mini Purification Kit For purification of PCR products, 100 bp to 20 kb www.tiangen.com TIANquick Mini Purification Kit (Spin column) Cat no. DP203 Kit Contents Contents Buffer BL Buffer PB Buffer

More information

Tamsulosin Hydrochloride Capsules

Tamsulosin Hydrochloride Capsules . nal Revision Bulletin Official October 1, 2011 Tamsulosin 1 standard solution, and shake well. Centrifuge at 1500 rpm for 10 min, and use the supernatant, passing it if Tamsulosin Hydrochloride Capsules

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

DNA Assembly and Enzymatic Cutting in Solutions: A Gold Nanoparticle Based SERS Detection Strategy

DNA Assembly and Enzymatic Cutting in Solutions: A Gold Nanoparticle Based SERS Detection Strategy Supporting Information DNA Assembly and Enzymatic Cutting in Solutions: A Gold Nanoparticle Based SERS Detection Strategy Elizabeth Crew 1, Hong Yan 1, Liqin Lin 1, Jun Yin 1, Zakiya Skeete 1, Timur Kotlyar

More information

Removal of Beta-Glucuronidase Enzyme from Urine Post-Hydrolysis. and Column Lifetime

Removal of Beta-Glucuronidase Enzyme from Urine Post-Hydrolysis. and Column Lifetime Removal of Beta-Glucuronidase Enzyme from Urine Post-Hydrolysis to Improve Assay Performance and Column Lifetime Shahana Huq, Seyed Sadjadi, and Sky Countryman Phenomenex, Inc., 411 Madrid Ave., Torrance,

More information

Western Blotting. Prepare samples:

Western Blotting. Prepare samples: Western Blotting Sive Lab Protocol March 2007 Prepare samples: For zebrafish embryos: Option 1: Take live embryos and put into 1.5 ml tube with E3. Centrifuge gently for 1-2 minutes -yolk lipids will rise

More information

Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C

Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C I. Introduction: Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N- phosphotransferase

More information

Protein Precipitation Protocols

Protein Precipitation Protocols Protein Precipitation Protocols Notes: All reagents need to high purity/hplc quality. All tubes used should be new or hand cleaned thoroughly with Micro90 detergent. High quality water needs to be used

More information

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Introduction Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical

More information

Malachite Green Phosphate Assay Kit

Malachite Green Phosphate Assay Kit Malachite Green Phosphate Kit Item No. 10009325 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions

More information

SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURES SUPPLEMENTARY FIGURES Fig. S1: Effect of ISO- and TAC-treatments on the biosynthesis of FAS-II elongation products in M. tb H37Ra. LC/MS chromatograms showing a decrease in products with elemental compositions

More information

The Automated SPE Assay of Fipronil and Its Metabolites from Bee Pollen.

The Automated SPE Assay of Fipronil and Its Metabolites from Bee Pollen. The Automated SPE Assay of Fipronil and Its Metabolites from Bee Pollen. Mark Crawford Single Presenter Name Abstract With the recent decline in hive bee populations this year the investigation of acaricides

More information

METHOD USED TO EXTRACT TOTAL MUSCLE PROTEIN FOR WESTERN BLOT USING TRIS-EDTA BUFFER*

METHOD USED TO EXTRACT TOTAL MUSCLE PROTEIN FOR WESTERN BLOT USING TRIS-EDTA BUFFER* METHOD USED TO EXTRACT TOTAL MUSCLE PROTEIN FOR WESTERN BLOT USING TRIS-EDTA BUFFER* SOLUTIONS FOR SAMPLE EXTRACTION 1. Tris-EDTA Buffer, ph 8.3 1 liter 50 mm Tris 6.06 g 10 mm EDTA 3.72 g Adjust ph to

More information

AxyPrep TM Mag PCR Clean-up Protocol

AxyPrep TM Mag PCR Clean-up Protocol AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR

More information

RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit

RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit User Manual Version 1.0 May 28, 2014 RayBio Creatine Kinase Activity Colorimetric Assay (Cat#: 68CL-CK-S100) RayBiotech, Inc. We Provide You

More information

Frozen-EZ Yeast Transformation II Catalog No. T2001

Frozen-EZ Yeast Transformation II Catalog No. T2001 INSTRUCTIONS Frozen-EZ Yeast Transformation II Catalog No. T2001 Highlights High transformation efficiency that yields approximately 10 5-10 6 transformants per μg plasmid DNA (circular). Frozen storage

More information

Genolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products

Genolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products Genolution Pharmaceuticals, Inc. Revolution through genes, And Solution through genes. Life Science and Molecular Diagnostic Products www.genolution1.com TEL; 02-3010-8670, 8672 Geno-Serum Hepatitis B

More information

Fighting the Battles: Conducting a Clinical Assay

Fighting the Battles: Conducting a Clinical Assay Fighting the Battles: Conducting a Clinical Assay 6 Vocabulary: In Vitro: studies in biology that are conducted using components of an organism that have been isolated from their usual biological surroundings

More information

Analysis of Casein and Whey Protein in Whole, 2%, and Skim Milk by Capillary Gel Electrophoresis

Analysis of Casein and Whey Protein in Whole, 2%, and Skim Milk by Capillary Gel Electrophoresis Analysis of Casein and Whey Protein in Whole, 2%, and Skim Milk by Capillary Gel Electrophoresis Marcia Santos, Staff Applications Scientist, Beckman Coulter Life Sciences, Brea, CA USA Mark Lies, Marketing

More information

Application of a New Immobilization H/D Exchange Protocol: A Calmodulin Study

Application of a New Immobilization H/D Exchange Protocol: A Calmodulin Study Application of a New Immobilization H/D Exchange Protocol: A Calmodulin Study Jiang Zhao; Mei Zhu; Daryl E. Gilblin; Michael L. Gross Washington University Center for Biomrdical and Bioorganic Mass Spectrometry:

More information

I. ACID-BASE NEUTRALIZATION, TITRATION

I. ACID-BASE NEUTRALIZATION, TITRATION LABORATORY 3 I. ACID-BASE NEUTRALIZATION, TITRATION Acid-base neutralization is a process in which acid reacts with base to produce water and salt. The driving force of this reaction is formation of a

More information

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Catalog Number. CSB-E14405r For the quantitative determination of rat creatine kinase MM isoenzyme (CK-MM) concentrations in serum, plasma and tissue

More information

Storage: Varies (see Kit Contents) Protocol part no. O r1 (MAN ) Rev. date: 24 April Introduction... 1 Kit Contents...

Storage: Varies (see Kit Contents) Protocol part no. O r1 (MAN ) Rev. date: 24 April Introduction... 1 Kit Contents... Vivid CYP450 Screening Kits User Guide Catalog no. P2856, P2857, P2858, P2859, P2860, P2861, P2862, P2863, P2864, P2969, P2970, P2972, P3019, P3021, PV6140, PV6141, and PV6142 Shipping: Varies Storage:

More information

USP Method Transfer of Donepezil Tablets from HPLC to UPLC

USP Method Transfer of Donepezil Tablets from HPLC to UPLC USP Method Transfer of Donepezil Tablets from HPLC to UPLC Mia Summers and Kenneth J. Fountain Waters Corporation, 34 Maple St., Milford, MA, USA APPLICATION BENEFITS Updating USP Methods from HPLC to

More information

In vitro analysis of pri-mirna processing. by Drosha-DGCR8 complex. (Narry Kim s lab)

In vitro analysis of pri-mirna processing. by Drosha-DGCR8 complex. (Narry Kim s lab) In vitro analysis of pri-mirna processing by Drosha-DGCR8 complex (Narry Kim s lab) 1-1. Preparation of radiolabeled pri-mirna transcript The RNA substrate for a cropping reaction can be prepared by in

More information

Automated Fast-Bead Synthesis of Small Peptides

Automated Fast-Bead Synthesis of Small Peptides Automated Fast-Bead Synthesis of Small Peptides Application Note 228 Joan Stevens, Ph.D., Norbert Wodke, Tim Hegeman and Kirby Reed (Gilson, Inc.) Introduction In proteomic research, the synthesis of peptides

More information

EdU Flow Cytometry Kit. User Manual

EdU Flow Cytometry Kit. User Manual User Manual Ordering information: (for detailed kit content see Table 2) EdU Flow Cytometry Kits for 50 assays: Product number EdU Used fluorescent dye BCK-FC488-50 10 mg 6-FAM Azide BCK-FC555-50 10 mg

More information

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION In this laboratory we will purify chloroplasts from spinach by differential centrifugation, then

More information

Fast, Reproducible LC-MS/MS Analysis of Dextromethorphan and Dextrorphan

Fast, Reproducible LC-MS/MS Analysis of Dextromethorphan and Dextrorphan Fast, Reproducible LC-MS/MS Analysis of and Kimberly Phipps, Thermo Fisher Scientific, Runcorn, Cheshire, UK Application Note 685 Key Words Accucore C18, dextromethorphan, dextrorphan, SOLA CX Abstract

More information

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280.

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. Technical Bulletin Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. PRINTED IN USA. Revised 4/06 AF9TB141 0406TB141 Wizard DNA Clean-Up System All technical literature is available on

More information

ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit

ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit Instructions for Use To quantify the cytotoxic effect of immune effector cells at a single cell level. This product is for research use only and

More information

TECHNICAL REPORT STUDY CHEMICAL PROPERTIES OF RATTAN SHOOT FROM PLANTATION IN THAILAND

TECHNICAL REPORT STUDY CHEMICAL PROPERTIES OF RATTAN SHOOT FROM PLANTATION IN THAILAND TECHNICAL REPORT STUDY CHEMICAL PROPERTIES OF RATTAN SHOOT FROM PLANTATION IN THAILAND by Assistant Professor Dr. Noojaree Prasitpan Chemist Analyzer of the ITTO Project on PD 24/00 Rev.1 (I): Promotion

More information

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS EXPERIMENT VI PURIFICATION AND CHARACTERIZATION OF PROTEINS I- Protein isolation and dialysis In order to investigate its structure and properties a protein must be obtained in pure form. Since proteins

More information

TECHNICAL BULLETIN. FluoroTag FITC Conjugation Kit. Product Number FITC1 Storage Temperature 2 8 C

TECHNICAL BULLETIN. FluoroTag FITC Conjugation Kit. Product Number FITC1 Storage Temperature 2 8 C FluoroTag FITC Conjugation Kit Product Number FITC1 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The FluoroTag FITC Conjugation Kit is suitable for the conjugation of polyclonal and

More information

Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit

Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit Catalog No. CSB-E15852c (96T) This immunoassay kit allows for the in vitro quantitative determination of canine CK-MB concentrations in serum and plasma.

More information

Creatine Kinase Microplate Assay Kit User Manual

Creatine Kinase Microplate Assay Kit User Manual Creatine Kinase Microplate Assay Kit User Manual Catalog # CAK1045 Detection and Quantification of Creatine Kinase (CK) Activity in Urine, Serum, Plasma, Tissue extracts, Cell lysate, Cell culture media

More information

Amaxa Mouse T Cell Nucleofector Kit

Amaxa Mouse T Cell Nucleofector Kit Amaxa Mouse T Cell Nucleofector Kit For T cells isolated from C57BL/6 & BALB/c mice Evaluated for murine T cells isolated from C57BL/6 & BALB/c mice This protocol is designed for murine lymphocytes or

More information

Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE EXPRESS WCX Prior to LC-MS/MS Analysis

Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE EXPRESS WCX Prior to LC-MS/MS Analysis Application Note AN844 Extraction of, and from Human Plasma Using EVOLUTE EXPRESS WCX Page 1 Extraction of, and from Human Plasma Using EVOLUTE EXPRESS WCX Prior to LC-MS/MS Analysis Introduction Catecholamines

More information

Islet Viability Assessment by Single Cell Flow Cytometry

Islet Viability Assessment by Single Cell Flow Cytometry Islet Viability Assessment by Single Cell Flow Cytometry Page 1 of 8 Purpose: To comprehensively assess the viability of the islet cell preparation prior to transplantation. Tissue Samples: A sample containing

More information

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,

More information

QuickZyme Soluble Collagen Assay

QuickZyme Soluble Collagen Assay QuickZyme Soluble Collagen Assay Version June 2012 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES This package insert must be read in its entirety before using this product. Introduction Collagen

More information

ACID-BASE TITRATIONS: DETERMINATION OF CARBONATE BY TITRATION WITH HYDROCHLORIC ACID BACKGROUND

ACID-BASE TITRATIONS: DETERMINATION OF CARBONATE BY TITRATION WITH HYDROCHLORIC ACID BACKGROUND #3. Acid - Base Titrations 27 EXPERIMENT 3. ACID-BASE TITRATIONS: DETERMINATION OF CARBONATE BY TITRATION WITH HYDROCHLORIC ACID BACKGROUND Carbonate Equilibria In this experiment a solution of hydrochloric

More information

Optimized Protocol sirna Test Kit for Cell Lines and Adherent Primary Cells

Optimized Protocol sirna Test Kit for Cell Lines and Adherent Primary Cells page 1 of 8 sirna Test Kit for Cell Lines and Adherent Primary Cells Kit principle Co-transfection of pmaxgfp, encoding the green fluorescent protein (GFP) from Pontellina p. with an sirna directed against

More information

For Research Use Only Ver

For Research Use Only Ver INSTRUCTION MANUAL Quick-RNA MiniPrep Catalog Nos. R1054 & R1055 Highlights High-quality total RNA (including small RNAs) from a wide range of samples. You can opt to isolate small and large RNAs in separate

More information

Laboratory Math II: Solutions and Dilutions

Laboratory Math II: Solutions and Dilutions Slide 1 Laboratory Math II: Solutions and Dilutions Philip Ryan, PhD Post-Doctoral Fellow National Cancer Institute, NIH Welcome to the National Institutes of Health, Office of Intramural Training & Education

More information

Protein Kinase Assay Kit, Universal Cat. No

Protein Kinase Assay Kit, Universal Cat. No User Protocol 539551 Rev. 16-September-04 JSW Page 1 of Protein Kinase Assay Kit, Universal Cat. No. 539551 Introduction Protein kinases catalyze the transfer of gamma phosphate from adenosine triphosphate

More information

THE FOLLOWING TEXT ON BIO-OIL EXTRACTION IS AN EXCERPT FROM THE FINAL TECHNICAL REPORT SUBMITTED BY HNEI TO OFFICE OF NAVAL RESEARCH UNDER GRANT

THE FOLLOWING TEXT ON BIO-OIL EXTRACTION IS AN EXCERPT FROM THE FINAL TECHNICAL REPORT SUBMITTED BY HNEI TO OFFICE OF NAVAL RESEARCH UNDER GRANT THE FOLLOWING TEXT ON BIO-OIL EXTRACTION IS AN EXCERPT FROM THE FINAL TECHNICAL REPORT SUBMITTED BY HNEI TO OFFICE OF NAVAL RESEARCH UNDER GRANT AWARD NUMBER N00014-09-1-0709, DECEMBER 2011 2.4 Novel Solvent-Based

More information

Protocol for Western Blotting

Protocol for Western Blotting Protocol for Western Blotting Materials Materials used on Day 3 Protease inhibitor stock: 1 μg/μl pepstatin A in DMSO 200 μm leupeptin in OG Buffer 200 mm PMSF: Freshly made. Ex) 34.8 mg PMSF in 1 ml isopropanol

More information

Solid-phase Synthesis of Homodimeric Peptides: Preparation of Covalently-linked Dimers of Amyloid-beta Peptide

Solid-phase Synthesis of Homodimeric Peptides: Preparation of Covalently-linked Dimers of Amyloid-beta Peptide Electronic Supplementary Information Solid-phase Synthesis of Homodimeric Peptides: Preparation of Covalently-linked Dimers of Amyloid-beta Peptide W. Mei Kok, a,b,c Denis B. Scanlon, b John A. Karas,

More information

Glycolysis Cell-Based Assay Kit

Glycolysis Cell-Based Assay Kit Glycolysis Cell-Based Assay Kit Item No. 600450 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION

More information

ab185915 Protein Sumoylation Assay Ultra Kit

ab185915 Protein Sumoylation Assay Ultra Kit ab185915 Protein Sumoylation Assay Ultra Kit Instructions for Use For the measuring in vivo protein sumoylation in various samples This product is for research use only and is not intended for diagnostic

More information

Automated Solid Phase Extraction (SPE) of EPA Method 1694 for Pharmaceuticals and Personal Care Products in Large Volume Water Samples

Automated Solid Phase Extraction (SPE) of EPA Method 1694 for Pharmaceuticals and Personal Care Products in Large Volume Water Samples Automated Solid Phase Extraction (SPE) of EPA Method 1694 for Pharmaceuticals and Personal Care Products in Large Volume Water Samples Keywords Application Note ENV0212 This collaboration study was performed

More information

Isolation. mircury Exosome Isolation Kit Serum and plasma

Isolation. mircury Exosome Isolation Kit Serum and plasma Isolation mircury Exosome Isolation Kit Serum and plasma Instruction manual v1.2 #300101 October 2015 Table of Contents Product summary......................................................... mircury

More information

Testosterone EIA Kit User Manual

Testosterone EIA Kit User Manual B-Bridge International, Inc. Testosterone EIA Kit User Manual Catalog# K3032-1 1 Plate Kit K3032-5 5 Plate Kit 1 TABLE OF CONTENTS Intended Use 3 Background 3 Assay Principle 3 Kit Components 4 Materials

More information

UltraClean PCR Clean-Up Kit

UltraClean PCR Clean-Up Kit UltraClean PCR Clean-Up Kit Catalog No. Quantity 12500-50 50 Preps 12500-100 100 Preps 12500-250 250 Preps Instruction Manual Please recycle Version: 02212013 1 Table of Contents Introduction... 3 Protocol

More information

Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit

Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Catalog No: E0479r 96 Tests Operating instructions www.eiaab.com FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH

More information

Section 3: Factors That Affect the Rate of Enzyme Catalyzed Reactions best

Section 3: Factors That Affect the Rate of Enzyme Catalyzed Reactions best Biology 12 Name: Metabolism Practice Test Section 1: What is an enzyme? 1. Which of the following statements is true about enzymes? a) 3D shape can vary and still be active b) they may catalyze only 1

More information

Opiates in Urine by SAMHSA GC/MS

Opiates in Urine by SAMHSA GC/MS application Note Gas Chromatography/ Mass Spectrometry Author Timothy D. Ruppel PerkinElmer, Inc. Shelton, CT 06484 USA Opiates in Urine by SAMHSA GC/MS Introduction The United States Department of Health

More information

Supporting Information

Supporting Information Supporting Information Wiley-VCH 2005 69451 Weinheim, Germany Magnetic Nanoparticle-Capped Mesoporous Silica Nanorod-Based Stimuli-Responsive Controlled Release Delivery System** Supratim Giri, Brian G.

More information