CHRISTIAN LAB WESTERN BLOT PROTOCOL

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "CHRISTIAN LAB WESTERN BLOT PROTOCOL"

Transcription

1 CHRISTIAN LAB WESTERN BLOT PROTOCOL There is actually 2 parts to a western blot: A. SDS-PAGE: Separates protein by size. Smaller proteins migrate faster through the gel than larger proteins. Size separation is most accurate in the middle of the gel. Higher gel percentages will separate smaller proteins and lower gel percentages will separate larger proteins (see Appendix A). B. Immunoblot: The proteins that have been separated by SDS-PAGE have been transferred to a nitrocellulose membrane, thus maintaining their relative positions. We can now determine the size and relative amount of our specific protein using an antibody raised against that protein. To visualize the interaction, a secondary antibody is used that is coupled to horse raddish peroxidase (HRP). In presence of a chemiluminescent substrate, HRP generates a light signal which can be detected with X-ray film or using a specialized camera. Day 1: prepare samples and/or pour gels (2h) Day 2: (pour gels) run gels, transfer to nitrocellulose, blocking & add primary Ab (4-5h) Day 3: develop western blot (2h) A. SDS-PAGE: 1. PREPARE SAMPLES MATERIALS (see Appendix B for recipes) Lysis Buffer: the one you choose depends on what type of protein you are looking at and how strong you need the lysis buffer to be Examples of possible choices: 1% Triton-100 LB, RIPA LB Protease and Phosphatase Inhibitors 1. phenylmethylsulfonyl fluoride (PMSF): protease inhibitor,10mg/ml in isopropanol, use at 1/ Aprotinin (protease inhibitor): stock 1000X 3. Leupeptin (Leu): stock at 1mg/ml, use at 1/ Sodium OrthoVanadate (Van): tyrosine phosphatase inhibitor, stock at 100 mm, use at 1/100 5X Sample buffer (SB) Working solutions Lysis buffer + inhibitors (LB+I) make up enough lysis buffer containing protease and phosphatase inhibitors (see above) for all your samples + 10% 1X Sample buffer dilute 5X sample buffer in water to make 1X sample buffer S.L. Christian September 2008 edited: Feb 2011 page. 1

2 PROCEDURE 1. Lyse cells in appropriate LB+I 2. Determine protein concentration using BCA assay (Appendix C) 3. Make up samples to contain 1X SB and equal protein concentrations up to the same total volume (see below for example) Concentration Volume cell Volume 3X BPB Total (µg/µl) lysate (µl) H 2 0 (µl) (µl) X=7 µg 14- X µl 7 µl 21 µl Example µl 1. incubate at 100 C (ie. boil) for 5 min. 2. Let samples cool for a minute, then do quick spin in centrifuge to get all liquid to bottom of tube. 3. Make up standards to same volume as samples (eg. 15 µl of sample buffer + 4 µl of prestained standards) 2. POUR GELS Note: Acrylamide is toxic, wear gloves at all times! MATERIALS SDS-PAGE Separating Gel Recipes Makes 7 ml= 2 gels 6% 8% 10% 12% 15% dh 2 O 993 µl x µl x 4 3 ml 800 µl x µl x M Tris 937 µl x µl x µl x µl x µl x 2 ph % 750 µl x µl x µl x µl x µl x 4 Acrylamide (29:1) 10% SDS % APS TEMED SDS-PAGE Stacking Gel Recipe 4.5% ; 3 ml per 2 gels dh 2 O 860 µl x M Tris ph µl 30% Acrylamide 500 µl 10% SDS 30 µl 10% APS 30 µl TEMED 3 µl S.L. Christian September 2008 edited: Feb 2011 page. 2

3 PROCEDURE Assembling plates and Pouring Gels 1. Acrylamide and 10% APS at 4 C, everything else at RT. 2. Wearing gloves, wash front gel plate (has notch) and back gel plate (no notch but has rounded corners) with dilute 7X and scrubber. Rinse extensively with distilled water and dry. Lay plates down on paper towel. 3. Rinse and dry 3 spacers. Make sure they are all the same size. 4. Wash the plates with 100% EtOH and wipe with kimwipes. 5. Take the back plate and wrap a yellow (0.75 mm) gel wrap around it. The breaks in the gel wrap should correspond to where the corners are. 6. Lay the plate down on the table and place spacers against the left and right edges. The rounded part of the spacer should fit into the rounded corner. Make sure the spacer touches the gel wrap but it is important that it the spacer is not under the gel wrap (this will cause leaks). 7. Carefully lay the top plate (clean side down) onto the bottom plate. 8. Pick up the two plates and square them up. Use the extra spacer to push one of the spacers back into place, if necessary. Put two clamps over the spacer (be sure to use the clamps that close all the way; the ones with wider openings are for assembling the plates into the gel box). 9. Push the other spacer back into the correct position and place two clamps over it. 10. Put two clamps on the bottom side and stand up the assembled gel plates. 11. Use the level to make sure the top of the plates are level. 12. Make a mark cm below the notch in the front plate. Pouring Separating Gel 1. Recipes for gels with different % acrylamide are listed in MATERIALS section. Add the reagents in the order indicated. 2. Pipet the separating gel mix between the plates up to the mark. Touching the pipet to the back plate will ensure smooth pouring and avoid bubbles. 2. Using a P-1000, gently overlay the acrylamide mix with ml of isopropanol. 3. Let gel polymerize for about an hour. 4. You will need the APS again for the stacking gel, so put it in the fridge (good for ~1 week). Pouring Stacking Gel 1. Get the APS and acrylamide from fridge. 2. Find the appropriate comb to make the wells for the gel and rinse it off. Make sure it will fit between the spacers on your gel. 3. Pour off the isopropanol from the top of the gel. (blot dry with Whatman paper) 4. Rinse the top of the gel with distilled water from squirt bottle and pour this off. 5. Tilt gel onto one corner and let it any remaining fluid drain to one side while you make the stacking gel mixture. 6. Add first three reagents of stacking gel mixture in a small glass flask. 7. Use a piece of 3MM paper to remove the last bit of liquid from the top of the gel. 8. Add the APS and the TEMED to the stacking gel mix and pipet the stacking gel mix between the gel plates to within a few mm of the top of the notch. 9. Insert the spacer into the stacking gel mix, trying not to get any bubbles beneath the teeth. It is ok if extra acrylamide spills over the edge. Do this on a paper towel. Work quickly because the stacking gel will polymerize within a few minutes. S.L. Christian September 2008 edited: Feb 2011 page. 3

4 10. If necessary, pipet a bit of stacking gel mix around the sides of the spacers to bring the level back up to the top of the notch. 3. LOAD SAMPLES, RUN GEL, TRANSFER TO NITROCELLUOSE MATERIALS 10X Running Buffer 1X Running Buffer 60.6 g Tris 200 ml 10X Running Buffer 288 g Glycine 1800 ml dh 2 O 20 g SDS Adjust to 2 L with dh 2 O Transfer Buffer (~1L/transfer box) 2.9 g Glycine 5.8 g Tris base add water to 800 ml, stir until dissolved 200 ml Methanol PROCEDURE 1. Put gel into apparatus for running gels 2. Pour 1X running buffer into upper and lower chambers 3. Carefully remove comb, running buffer will flow into wells and rinse out unpolymerized acrylamide 4. If running only one gel, clamp spacer plate on the other side. 5. Load the samples using gel loading tips. Place the tip close to the bottom of the well. The sample is denser than the running buffer and will sink to the bottom of the well. Be careful not to expel a big air bubble at the end and force sample out of the well. 6. load equal volume of 1X sample buffer in all empty lanes 7. When all samples are loaded, place the cover on the gel box matching color of electrodes. 8. Turn on the power supply. Gel Running Conditions Run gel at 100V until dye front is near the bottom (1 2h). (Optional: For overnight, run at V. In the morning, turn up the power to 100 V for a while to refocus the bands.) 1. Turn off the power supply. 2. Place the gel box in a pan or sink. 3. Undo the clamps and remove the gel plates. 4. Using a spacer, pry apart the plates. The gel should stick to one plate or the other. 5. If you are going to do a transfer, you should have already set up the transfer cassette. 6. Turn the plate upside down over the transfer cassette, prod the gel with the spacer and let it fall into the buffer. Mini-gel transfer to nitrocellulose 1. While gel is running: a) Make transfer buffer. b) Get pan for assembling sandwich and rinse with distilled water. S.L. Christian September 2008 edited: Feb 2011 page. 4

5 c) Get transfer box, cassettes, pads and rinse with distilled water. d) Get filter forceps and rinse. e) Get 4 pieces of 3MM filter paper per gel (6 x 9 cm) f) Get 1 nitrocellulose membrane per gel (6 x 8 cm). Equilibrate in transfer buffer for 30 minutes before transfer. g) Fill pan with transfer buffer. h) Place cassette in pan with black side down. i) Soak pad in transfer buffer and place on cassette. 2. Assemble sandwich-wear gloves! a) Soak 2 pieces 3MM paper and place on pad. b) Place gel on 3MM paper. Gel should be face down so that blot comes out with same orientation as loaded. Smooth out with fingers. c) Label upper right hand side of nitrocellulose with pencil to maintain orientation. Use forceps to place nitrocellulose on gel. Run pipette over it to get rid of bubbles. d) Place 2 pieces of 3MM on nitrocellulose. Smooth out with pipette. e) Soak pad and place on top. f) Close cassette and put in transfer box with black side of cassette facing the black side of the transfer box insert. g) Put white plastic insert with ice into transfer box. h) Fill transfer box with transfer buffer. 3. Run at 70 volts for 75 min. Starting current = 0.19A for two gels. OR Run at 100 volts for 1h with stirring (optional: To transfer overnight, run at V. Omit ice insert) B. IMMUNOBLOT MATERIALS 10X TBS (Tris buffered saline) g NaCl 48.5 g Tris base Adjust volume to 2L ph to 7.5 (add approx. 29ml conc. HCl) TBST (0.05) 2L 1L 10X TBS 200 ml 100 ml water 1800 ml 900 ml 10% Tween ml 5 ml PROCEDURE 1. Remove nitrocellulose membrane from cassette (discard gel and filter papers) 2. Block with 20 ml 5% milk/tbst or 10 ml 5% BSA/TBS, as appropriate for antibody used, for 1 h at RT with rocking 3. Add primary antibody ON at 4 C with rocking 4. Wash min, 3 changes, with TBST S.L. Christian September 2008 edited: Feb 2011 page. 5

6 5. Add appropriate secondary antibody (HRP conjugate) 1hr at RT with shaking (1:5000 in TBST) 6. Wash min, 3 changes, with TBST 7. Prepare Chemiluminescent Substrate, mix 2 ml Reagent A plus 2 ml Reagent B 8. Incubate membrane 5 min at RT 9. blot corners of membrane on filter paper 10. wrap in SaranWrap, tape in autorad cassette 11. expose to camera and take picture Strip & Reprobe (optional if you want to reprobe the same membrane with a different Ab) 1. Rinse membrane with TBS 2. wash 3 x 10 min with TBS ph 2.0 (100 ml 10XTBS, 900 ml dh 2 O, 1.86ml conc HCl) 3. rinse with TBS 4. block 1h with appropriate blocking buffer 5. Add primary antibody ON at 4 C with rocking 6. follow step 5-12 above S.L. Christian September 2008 edited: Feb 2011 page. 6

7 APPENDIX A From the BioRad Catalogue S.L. Christian September 2008 edited: Feb 2011 page. 7

8 APPENDIX B 1% Triton X-100 lysis buffer- Store at 4 C Reagent Final concentration For 500 ml, use Water up to 500 ml 1 M TrisHCl, ph 8 20 mm 10 ml 10% Tx-100 1% 50 ml glycerol 10% 100 ml of 50% (w/v) in water EDTA 2 mm 2 ml of 0.5 M, ph 8.0 NaCl 137 mm 13.7 ml of 5M RIPA-Tris based Final Stock Volume 50 mm Tris-HCl (ph 7.6) 1M 25 ml 5 ml 0.02% sodium azide 10% 1 ml 200 µl 0.5% sodium deoxycholate 10% 25 ml 5ml 0.1% SDS 10% 5 ml 1 ml 1% NP-40 10% 50 ml 10 ml 150 mm NaCl 5 M 15 ml 3 ml H20 up to 500 ml up to 100 ml RIPA- Phosphate based Final Stock Volume 1X PBS 10X 50 ml 10 ml 0.5% sodium deoxycholate 2.5g 0.5g 0.1% SDS 10% 50 ml 10 ml 1% NP-40 10% 50 ml 10 ml H20 up to 500 ml up to 100 ml 5X SDS-PAGE Sample Buffer with 1X Glycerol (20 ml) mm Tris base g or M Tris, ph % Glycerol 2 g 11.5% SDS 2.3 g 500 mm DTT 1.54 g (Omit for Non-Reducing Gels) ph to 6.8 Adjust volume to 20 ml 0.1% Bromophenol Blue 20 mg (0.020 g) (Add last after phing) Make 1 ml aliquots and store at 20 C S.L. Christian September 2008 edited: Feb 2011 page. 8

9 APPENDIX C BCA protein assay From Pierce, includes 2 mg/ml BSA standard 1. Set up 1.5 ml µfuge tubes with standards or samples in final volume of 50 ul. Standards should receive same amount of lysis buffer as sample tubes. Example: Tube # µl Water µl Lysis buffer µl 2 mg/ml BSA in µg BSA in µg/µl BSA water assay Samples µl sample 2. All standards should get same volume of identical lysis buffer used for samples. If assaying a different sample volume, adjust amount of lysis buffer added to standards and increase or decrease volume of water accordingly. 3. Mix Pierce BCA reagent: 50 parts soln A + 1 part soln B. 4. Add 1 ml to each sample. Vortex. 5. Standard protocol: 30 minutes in 37 o water bath. Enhanced protocol: 30 min in 60 C water bath 6. Vortex, cool 5 minutes. 7. Read A562 vs tube #1 as blank. Read all samples within 10 minutes. Use quartz cuvettes. For standards, just empty cuvette between each as you go in ascending order. Then wash out with water and do samples. 8. To calculate protein concentrations of samples, plot ug/ul BSA std (Y axis) vs OD562 (X axis). Do curve fit and get linear equation. Y = µg protein. Take ODs of samples (X) and plug into equation. S.L. Christian September 2008 edited: Feb 2011 page. 9

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

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

Running protein gels and detection of proteins

Running protein gels and detection of proteins Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.

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

Western Blotting: Mini-gels

Western Blotting: Mini-gels Western Blotting: Mini-gels Materials a Protein Extraction Buffer (for callus or kernel), Solution Stock Final Volume Tris-HCl ph 80 1 M 200 mm 20 ml NaCl 4 M 100 mm 25 ml Sucrose 2 M 400 mm 20 ml EDTA

More information

SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies

SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies Pouring the resolving gel 1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and

More information

Western Blotting For Protein Analysis

Western Blotting For Protein Analysis Western Blotting For Protein Analysis Part 1: Laemmli Gel Electrophoresis Using Mini-PROTEAN II Electrophoresis Cell Note: Powder-free gloves should be worn throughout the entire procedure. A. Preparing

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

Western Blot Protocol Protein isolation

Western Blot Protocol Protein isolation Western Blot Protocol Protein isolation A. Preparation of cell lysates. - Preparation of materials: -Dial the microcentrifuge temperature control setting to 4 C -Prepare a bucket of ice -Prepare lysis

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

2. Cut 6 sheets of Whatman 3MM paper and 1 sheet of blotting membrane to the size of the gel, or slightly smaller.

2. Cut 6 sheets of Whatman 3MM paper and 1 sheet of blotting membrane to the size of the gel, or slightly smaller. Method for Western Blotting Western Blotting ELECTROPHORESIS Prepare and run an SDS PAGE gel. Select a gel percent which will give the best resolution for the size of antigen being analyzed (if known).

More information

Western Blotting. USA: proteintech@ptglab.com UK & Europe: europe@ptglab.com China: service@ptglab.com. www.ptglab.com

Western Blotting. USA: proteintech@ptglab.com UK & Europe: europe@ptglab.com China: service@ptglab.com. www.ptglab.com Western Blotting All steps are carried out at room temperature unless otherwise indicated. Recipes for all solutions highlighted bold are included at the end of the protocol. SDS-PAGE 1. Construct an SDS-PAGE

More information

ECL Western Blotting Substrate INSTRUCTIONS FOR USE OF PRODUCTS W1001 AND W1015.

ECL Western Blotting Substrate INSTRUCTIONS FOR USE OF PRODUCTS W1001 AND W1015. Technical Manual ECL Western Blotting Substrate INSTRUCTIONS FOR USE OF PRODUCTS W1001 AND W1015. PRINTED IN USA. 6/09 ECL Western Blotting Substrate All technical literature is available on the Internet

More information

Product name Company Cat # PowerPac Basic Power supply Bio Rad 165-6019 Mini Protean electrophoresis system Mini trans blot cell Bio Rad 170-3930

Product name Company Cat # PowerPac Basic Power supply Bio Rad 165-6019 Mini Protean electrophoresis system Mini trans blot cell Bio Rad 170-3930 SDS-PAGE and western blot for low molecular weight proteins (2-20kDa) Merav Marom Shamur, Smart Assays Aim: Analysis of low molecular weight proteins by SDS-PAGE and western blot under reducing conditions.

More information

Western Blot Protocol. Add 7.5ml of res gel to plates; Add diwater (to make sure it s flat.)

Western Blot Protocol. Add 7.5ml of res gel to plates; Add diwater (to make sure it s flat.) Western Blot Protocol 1. Cast Gel: Assemble minigel apparatus( Be sure no leaking) Make resolution gel (recipe) and mix Make stacking gel (recipe) diwater acrylamide/bis Add 7.5ml of res gel to plates;

More information

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western).

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Western Blot SOP Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Date: 8/16/05, 10/31/05, 2/6/06 Author: N.Oganesyan, R. Kim Edited by: R. Kim Summary:

More information

STANDARD OPERATING PROCEDURE

STANDARD OPERATING PROCEDURE STANDARD OPERATING PROCEDURE Title: Evaluation using Western Blot SOP#: M-103 Version #: 1 Author: R. Saul Date Approved: Feb. 5, 2009 Date Modified: 1. PURPOSE The purpose of this document is to describe

More information

Western Blot Protocol (updated on 05/20/14)

Western Blot Protocol (updated on 05/20/14) Western Blot Protocol (updated on 05/20/14) Required Solutions 10x PBS (1L) 80 g NaCl 2 g KCl 14.4 g Na 2 HPO 4 or 22 g Na 2 HPO 4 7H 2 O 2.4 g KH 2 PO 4 or 2 g KH 2 PO4 Adjust ph to 7.4 Autoclave PBST

More information

SDS-PAGE and Western Blotting with the Odyssey Infrared Imaging System Entered by Kevin Janes Janes Lab Protocols 12/9/14

SDS-PAGE and Western Blotting with the Odyssey Infrared Imaging System Entered by Kevin Janes Janes Lab Protocols 12/9/14 I. SDS-PAGE: 1. Assemble glass sandwich a. Clean the short and 1.5 mm spacer plates with 70% alcohol before starting (one short and one spacer plate per gel). If there is residual polyacrylamide dried

More information

Southern Blot Analysis (from Baker lab, university of Florida)

Southern Blot Analysis (from Baker lab, university of Florida) Southern Blot Analysis (from Baker lab, university of Florida) DNA Prep Prepare DNA via your favorite method. You may find a protocol under Mini Yeast Genomic Prep. Restriction Digest 1.Digest DNA with

More information

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com PR110 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Dot Blot Analysis Teacher s Guidebook (Cat. # BE 502) think proteins! think G-Biosciences

More information

Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides

Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides Polyacrylamide gel electrophoresis (PAGE) and subsequent analyses are common tools in biochemistry and molecular biology. This Application

More information

PROTOCOL 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 www.mitosciences.com

PROTOCOL 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 www.mitosciences.com PROTOCOL Western Blotting Transfer and Detection Procedure 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 02-11 DESCRIPTION Western Blotting Transfer and Detection Procedure ADDITIONAL MATERIALS REQUIRED

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

WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TROUBLESHOOTING GUIDE

WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TROUBLESHOOTING GUIDE WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TIPS FOR SUCCESSFUL WESTERB BLOTS TROUBLESHOOTING GUIDE 1. Suboptimal protein transfer. This is the most common complaint with western blotting and could

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

2D gel electrophoresis

2D gel electrophoresis 2D gel electrophoresis Required solutions Cathode buffer: TRIS 7.6g Glycine 36g SDS 2.5g Fill up with ddwater to 250ml ESS (equilibration stock solution) SDS 2g Urea 36g EDTA 3mg 50 mm TRIS-HCl ph 6.8

More information

Biochemistry Lab SDS PAGE and Western blot General Instructions

Biochemistry Lab SDS PAGE and Western blot General Instructions Background When an electrical field is applied across a solution, the movement of the charged particles (proteins) is influenced not only by the charge but also the voltage, distance between electrodes,

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

Electrophoresis and Electroblotting of Proteins

Electrophoresis and Electroblotting of Proteins Electrophoresis and Electroblotting of Proteins The purpose of the next lab exercises will be to study the relative amounts of β-actin in cells of the B-16 melanoma, liver and muscle of mice. Electrophoresis

More information

Biology 309 Lab Notebook

Biology 309 Lab Notebook Name: Biology 309 Lab Notebook This is a guided lab notebook for you to keep well-organized notes about procedures and record experimental data for experiments as they are performed. It is guided because,

More information

Note: Tris-HCl Buffer is used for specific cases of immunohistochemical staining.

Note: Tris-HCl Buffer is used for specific cases of immunohistochemical staining. STOCK SOLUTION RECIPIES: Tris-HCl Buffer 10X Tris-HCl (0.5M Tris Base, ph7.6): Trizma Base ---------------------------------- 61 g Distilled water ------------------------------- 1000 ml Adjust ph 7.6

More information

30% Acrylamide 7.88 ml 11.38 ml 15.75 ml. Tris (ph 8.8) 4.5 ml 6.5 ml 9 ml. APS 135 ul 195 ul 270 ul. TEMED 6.75 ul 9.75 ul 13.

30% Acrylamide 7.88 ml 11.38 ml 15.75 ml. Tris (ph 8.8) 4.5 ml 6.5 ml 9 ml. APS 135 ul 195 ul 270 ul. TEMED 6.75 ul 9.75 ul 13. WESTERN BLOT Kyle Nvakwski Nvember 9 th, 2012 Bwdish Lab, McMaster University Hamiltn, ON, Canada www.bwdish.ca PROTOCOL Pre-wrk: Turn heat blck t 100C (If making lysates) Allw prtease inhibitr (Hpper,

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

2D gel Protocol. 2. Determining Protein Concentration of cell lysates

2D gel Protocol. 2. Determining Protein Concentration of cell lysates 2D gel Protocol 1. Lysis and Protein Extraction from cells Prepare cell lysates with Trizol extraction by following Kathleen Lyons s protocol: AfCS Procedure Protocol PP00000155, Version 1, 05/12/03 (Ref.1).

More information

Buffer Recipes For 2DE

Buffer Recipes For 2DE Buffer Recipes For 2DE Always use electrophoresis/molecular biology quality reagents or higher Always use 18.2 ohm water quality or higher (MilliQ or bottled) As indicated in individual buffers, some reagents

More information

Protocol #24 Western Blotting

Protocol #24 Western Blotting 1 of 5 Aim The aim of Western blotting is to enable the detection of protein via binding with an antibody against a recombinant tag or a natural epitope determinant on the surface of the protein. This

More information

Protein expression in the life cycle of bean beetles (Callosobruchus maculatus)

Protein expression in the life cycle of bean beetles (Callosobruchus maculatus) Protein expression in the life cycle of bean beetles (Callosobruchus maculatus) Pre laboratory Preparation Instructor s Notes You will need a number of cultures of bean beetles at various life stages.

More information

EZ-Run Protein Gel Solution. EZ-Run Protein Standards. EZ-Run Gel Staining Solution. Traditional SDS-Page Reagents. Protein Electrophoresis

EZ-Run Protein Gel Solution. EZ-Run Protein Standards. EZ-Run Gel Staining Solution. Traditional SDS-Page Reagents. Protein Electrophoresis EZ-Run Protein Gel Solution EZ-Run Protein Standards EZ-Run Gel Staining Solution Traditional SDS-Page Reagents Protein Electrophoresis protein electrophoresis Introduction Sodium dodecyl sulfate polyacrylamide

More information

CLONTECH. Antibodies Protocol Guide PT (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY. Innovative Tools to Accelerate Discovery

CLONTECH. Antibodies Protocol Guide PT (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY. Innovative Tools to Accelerate Discovery CLONTECH Innovative Tools to Accelerate Discovery Antibodies Protocol Guide PT3407-1 (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY Table of Contents I. Introduction 3 II. Materials Required

More information

WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB)

WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB) WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB) WESTERN BLOT FOR ANALYSIS ON LICOR ODYSSEY SCANNER. 1) The Licor Odyssey protein marker is optimal as it is visible on channel 700 (2ul is enough

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

Pure-IP Western Blot Detection Kit

Pure-IP Western Blot Detection Kit Product Manual Pure-IP Western Blot Detection Kit Catalog Number PRB-5002 20 blots FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The technique of immunoprecipitation (IP) is used

More information

WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots. Fluorescent detection via: Goat anti-mouse SureLight P3

WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots. Fluorescent detection via: Goat anti-mouse SureLight P3 WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots Fluorescent detection via: Goat anti-mouse SureLight P3 Cat. #: WK-P112 6440 Dobbin Road, Suite D Phone (443)

More information

Northern blot analysis for microrna. (Narry Kim s lab)

Northern blot analysis for microrna. (Narry Kim s lab) Northern blot analysis for microrna (Narry Kim s lab) Materials 1. 10~50 μg of total RNA extracted from HeLa cells treated with sirna 2. RNA loading buffer 3. Probe: DNA oligonucleotide complementary to

More information

Western BLoT Immuno Booster

Western BLoT Immuno Booster Cat. # T7111A For Research Use Western BLoT Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Storage... 3 IV. Materials Required but Not Provided... 3 V. Precautions... 3 VI.

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

AES Application Focus Blotting Page 1

AES Application Focus Blotting Page 1 AES Application Focus Blotting Page 1 Western Blotting Adapted from Chapter 7, Gel Electrophoresis of Proteins, by David E. Garfin, Pages 197-268, in Essential Cell Biology, Volume 1: Cell Structure, A

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

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

Lab 5: DNA Fingerprinting

Lab 5: DNA Fingerprinting Lab 5: DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the

More information

WESTERN BLOTTING - A BEGINNER S GUIDE

WESTERN BLOTTING - A BEGINNER S GUIDE WESTERN BLOTTING - A BEGINNER S GUIDE Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The blot is

More information

RAGE. Plugs for RAGE/PFGE

RAGE. Plugs for RAGE/PFGE 1 RAGE Rotating Field Gel Electrophoresis (RAGE) is a variation on Pulsed Field Gel Electrophoresis (PFGE) and gives similar results. We use equipment that was only briefly marketed by Stratagene, at far

More information

DNA Electrophoresis Lesson Plan

DNA Electrophoresis Lesson Plan DNA Electrophoresis Lesson Plan Primary Learning Outcomes: Students will learn how to properly load a well in an agarose gel. Students will learn how to analyze the results of DNA electrophoresis. Students

More information

Anti-ATF6 α antibody, mouse monoclonal (1-7)

Anti-ATF6 α antibody, mouse monoclonal (1-7) Anti-ATF6 α antibody, mouse monoclonal (1-7) 73-500 50 ug ATF6 (activating transcription factor 6) is an endoplasmic reticulum (ER) membrane-bound transcription factor activated in response to ER stress.

More information

Procedure for RNA isolation from human muscle or fat

Procedure for RNA isolation from human muscle or fat Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe

More information

Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution.

Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Introduction: The determination of protein concentration is frequently required in biochemical work. Several methods

More information

Technical Manual No. 0210 Update date 10112010

Technical Manual No. 0210 Update date 10112010 Express TM PAGE Gels Technical Manual No. 0210 Update date 10112010 I Description.. 1 II Gel Selection Guide. 1 III Protein Migration Table.. 2 IV Compatible Gel Boxes... 2 V Related Products. 3 VI Storage.

More information

Preparation of Parasite Protein Extracts and Western Blot Analysis Arlett Heiber and Tobias Spielmann *

Preparation of Parasite Protein Extracts and Western Blot Analysis Arlett Heiber and Tobias Spielmann * Preparation of Parasite Protein Extracts and Western Blot Analysis Arlett Heiber and Tobias Spielmann * Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany *For correspondence:

More information

Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA

Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA KAMIYA BIOMEDICAL COMPANY Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA For the quantitative determination of mouse CKMB in serum, plasma, cell culture fluid and other biological fluids Cat. No. KT-57681

More information

Compromise Elsewhere Protocols. Western Blotting Methods. 800-656-ROCK www.rockland-inc.com info@rockland-inc.com 1 of 11

Compromise Elsewhere Protocols. Western Blotting Methods. 800-656-ROCK www.rockland-inc.com info@rockland-inc.com 1 of 11 Compromise Elsewhere Protocols Western Blotting Methods info@rockland-inc.com 1 of 11 Odyssey Western Blotting Protocols Odyssey reagents: Additional reagents needed: IR-labeled secondary antibodies Odyssey

More information

For the development of sandwich ELISAs to measure phosphorylated Epidermal Growth Factor Receptor (EGF R) in cell lysates.

For the development of sandwich ELISAs to measure phosphorylated Epidermal Growth Factor Receptor (EGF R) in cell lysates. DuoSet IC Human Phospho-EGF R Catalog Number DYC1095-2 Catalog Number DYC1095-5 For the development of sandwich ELISAs to measure phosphorylated Epidermal Growth Factor Receptor (EGF R) in cell lysates.

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

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

Protein Detection Methods & Western Blots

Protein Detection Methods & Western Blots Protein Detection Methods & Western Blots 1.Sodium DodecylSlufate(SDS)-PolyacrylamideGel Electrophoresis (SDS-PAGE) Visualize many proteins at once 2. SDS-PAGE combined with immunoblotting (Western) Visualize

More information

Definitive guide to western blot

Definitive guide to western blot Definitive guide to western blot Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose

More information

Anti-V5 Antibody Anti-V5-HRP Antibody

Anti-V5 Antibody Anti-V5-HRP Antibody Anti-V5 Antibody Anti-V5-HRP Antibody Catalog nos. R960-25, R961-25 Version F 073001 28-0140 www.invitrogen.com tech_service@invitrogen.com ii Table of Contents Table of Contents...iii Overview...1 Western

More information

Fast Semi-Dry Transfer System with Yrdimes

Fast Semi-Dry Transfer System with Yrdimes Fast Semi-Dry Transfer System with Yrdimes INTRODUCTION Yrdimes is a semi-dry transfer system for Western blotting mainly used for fast and efficient blotting by using less transfer buffer. It is used

More information

A Guide to Protein Blotting

A Guide to Protein Blotting A Guide to Protein Blotting SDS-PAGE SDS-PAGE stands for sodium dodecyl (lauryl) sulphate-polyacrylamide gel electrophoresis. SDS-PAGE has a number of uses, which include: establishing protein size, protein

More information

Canine Creatine Kinase MM isoenzyme(ck-mm) ELISA. kit

Canine Creatine Kinase MM isoenzyme(ck-mm) ELISA. kit BlueGene Biotech. Tel: 0086-21-61471242 Fax: 0086-21-61471242 ext 806 E-mail: sales@bluegene.cc tech@bluegene.cc www.elisakit.cc www.bluegene.cc Canine Creatine Kinase MM isoenzyme(ck-mm) ELISA kit 96

More information

Western Blotting and ELISA Techniques

Western Blotting and ELISA Techniques Researcher, 2009;1(2):67-86 Yang and Ma, Western Blotting Method Western Blotting and ELISA Techniques Yan Yang *, Hongbao Ma *, ** * Brookdale University Hospital and Medical Center, Brooklyn, New York

More information

Protocol Micro Lab. K:\Microlab_protocols\Protocols_active\03 Instrument manuals\ml03_001_001 DGGE.doc. Preparation of gels

Protocol Micro Lab. K:\Microlab_protocols\Protocols_active\03 Instrument manuals\ml03_001_001 DGGE.doc. Preparation of gels Preparation of gels Cautions: Wear gloves all times when handling acryl amide and form amide solutions and all work with acryl amide and form amide has to be done in a flow hood. Acryl amide is very toxic.

More information

RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis

RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis INTRODUCTION This laboratory will demonstrate the basics of electrophoresis and the theory behind the separation of molecules on an agarose

More information

IMMUNOPRECIPITATION (IP) PROTOCOL

IMMUNOPRECIPITATION (IP) PROTOCOL IMMUNOPRECIPITATION (IP) PROTOCOL Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract so that the antibody

More information

APPLICATION FOCUS. Application Solutions for Western Blotting

APPLICATION FOCUS. Application Solutions for Western Blotting APPLICATION FOCUS Application Solutions for Western Blotting WESTERN BLOTTING Companion Products Solutions for consistently better blotting. Companion products from Cell Signaling Technology (CST) are

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

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

THE ACTIVITY OF LACTASE

THE ACTIVITY OF LACTASE THE ACTIVITY OF LACTASE Lab VIS-8 From Juniata College Science in Motion Enzymes are protein molecules which act to catalyze the chemical reactions in living things. These chemical reactions make up the

More information

Nitrotyrosine Western blot starter pack

Nitrotyrosine Western blot starter pack Nitrotyrosine Western blot starter pack Cat no. A010-513 A convenient reagent pack containing Badrilla s high integrity nitrotyrosine monoclonal antibody (clone 2E11) and associated positive and negative

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, tissue homogenates.

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

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

Mouse krebs von den lungen 6 (KL-6) ELISA

Mouse krebs von den lungen 6 (KL-6) ELISA KAMIYA BIOMEDICAL COMPANY Mouse krebs von den lungen 6 (KL-6) ELISA For the quantitative determination of mouse KL-6 in serum, plasma, cell culture supernatants, body fluid and tissue homogenate Cat. No.

More information

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2 Uscn Life Science Inc. Wuhan Website: www.uscnk.com Phone: +86 27 84259552 Fax: +86 27 84259551 E-mail: uscnk@uscnk.com ELISA Kit for Human Prostaglandin E1(PG-E1) Instruction manual Cat. No.: E0904Hu

More information

About the Kits...2 Description 2 Components 2

About the Kits...2 Description 2 Components 2 Table of Contents About the Kits...2 Description 2 Components 2 Thrombin Cleavage...3 Small scale optimization 3 Scale-up 4 Factors that affect thrombin activity 4 Monitoring cleavage 5 Biotinylated Thrombin

More information

The MiniOne TM Reagent Kit: Electrophoresis 101

The MiniOne TM Reagent Kit: Electrophoresis 101 The MiniOne TM Reagent Kit: Cat#: M3001 2 3 Reagent Kit Components Other Required Materials Laboratory Safety Experiment Procedures Teacher s Guide Student Worksheet Ordering Consumables Table of Contents

More information

竞 争 性 分 析 Epitope Mapping 实 验 方 法

竞 争 性 分 析 Epitope Mapping 实 验 方 法 竞 争 性 分 析 Epitope Mapping 实 验 方 法 ABSTRACT The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can

More information

Primary Antibody-polyHRP Labeling Western Blot Kit. Cat. No. A

Primary Antibody-polyHRP Labeling Western Blot Kit. Cat. No. A Primary Antibody-polyHRP Labeling Western Blot Kit Cat. No. A-9402-001 April 2011 I. Introduction Western Blot Detection Development of western blots with secondary antibody/hrp conjugates while simple

More information

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

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

Olympic B3 Summer Science Camp 2015 Weller, Smith, Putnam L3

Olympic B3 Summer Science Camp 2015 Weller, Smith, Putnam L3 Chestnut Leaf DNA Extraction Protocol Introduction: we will extract the nucleic acid from leaf tissue by grinding it in a reducing medium (the beta-mercaptoethanol chemical is a reducing agent, it smells

More information

(Obtained from John Stebbins, Triezenberg lab)

(Obtained from John Stebbins, Triezenberg lab) ASSAY OF β-galactosidase IN YEAST (Obtained from John Stebbins, Triezenberg lab) There are two basic methods for the in vitro assay of B-galactosidase activity from yeast. They differ mainly in the method

More information

HUMAN ANTERIOR GRADIENT 2 (AGR2) ELISA KIT

HUMAN ANTERIOR GRADIENT 2 (AGR2) ELISA KIT HUMAN AGR2 ELISA KIT P a g e 1 HUMAN ANTERIOR GRADIENT 2 (AGR2) ELISA KIT FOR THE QUANTITATIVE DETERMINATION OF HUMAN AGR2 CONCENTRATIONS IN CELL CULTURES, SERUM AND PLASMA. PURCHASE INFORMATION: ELISA

More information

Culturing Chick Embryonic Myoblasts/Myotubes and Fibroblasts

Culturing Chick Embryonic Myoblasts/Myotubes and Fibroblasts Culturing Chick Embryonic Myoblasts/Myotubes and Fibroblasts Day 1 1. 100 ug/ml gelatin (described separately later). 1. Pipet 10 ml gelatin onto each of two 100 mm culture dishes. Leave in sterile hood

More information

Standard Operating Procedure (SOP)

Standard Operating Procedure (SOP) Standard Operating Procedure (SOP) Title: Direct Fluorescent Antibody Test (DFAT) for the detection of Renibacterium salmoninarum in tissues Number: BACT-3 Version: 02 Created December 14, 2011 Approval:

More information

EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA

EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA Biochemistry Lab CHE 431 Page 1 of 7 OBJECTIVE: EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA DNA splicing, the cutting and linking of DNA molecules, is one of the basic tools of modern

More information

Mouse Testosterone ELISA

Mouse Testosterone ELISA KAMIYA BIOMEDICAL COMPANY Mouse Testosterone ELISA For the quantitative determination of mouse Testosterone in biological samples. Cat. No. KT-52637 For Research Use Only. Not for use in diagnostic procedures.

More information

Mini-PROTEAN Precast Gels

Mini-PROTEAN Precast Gels Mini-PROTEAN Precast Gels Instruction Manual and Application Guide For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-4BIORAD (1-800-424-6723) Table of Contents Section

More information

Factors Affecting Enzyme Activity

Factors Affecting Enzyme Activity INTRODUCTION Factors Affecting Enzyme Activity The chemical reactions occurring in living things are controlled by enzymes. An enzyme is a protein in the cell which lowers the activation energy of a catalyzed

More information

Biology 3460 - Plant Physiology - Lab Exercise 2 Basic Lab Techniques

Biology 3460 - Plant Physiology - Lab Exercise 2 Basic Lab Techniques Biol3460 Lab 1 1 Biology 3460 - Plant Physiology - Lab Exercise 2 Basic Lab Techniques Objectives: This lab is intended to: (1) provide an introduction to basic techniques used in biology labs (2) provide

More information