Anti-ACTIVE MAPK, p38 and JNK Polyclonal Antibodies and Anti-ACTIVE Qualified Secondary Antibody Conjugate

Transcription

1 Technical Bulletin Anti-ACTIVE MAPK, p38 and JNK Polyclonal Antibodies and Anti-ACTIVE Qualified Secondary Antibody Conjugate INSTRUCTIONS FOR USE OF PRODUCTS V1211, V8031, V7931, V7932 AND V7951. PRINTED IN USA. Revised 1/12.

2 Anti-ACTIVE MAPK, p38 and JNK Polyclonal Antibodies and Anti-ACTIVE Qualified Secondary Antibody Conjugate All technical literature is available on the Internet at Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use of this system. 1. Description Product Components and Storage Conditions General Considerations Western Blot Analysis with Anti-ACTIVE pabs...7 A. Probing Membranes with Anti-ACTIVE pabs...7 B. Detection Protocols Immunocytochemistry Protocol for Anti-ACTIVE pabs Composition of Buffers and Solutions References Related Products Description Promega has developed polyclonal antibodies (pab) to three members of the Mitogen-Activated Protein Kinase (MAPK) superfamily of enzymes. These enzymes, which play important roles in signal transduction in eukaryotic cells, are MAPK, p38 and JNK. These kinases have interconnected roles in a cascade of signal transduction reactions. The Anti-ACTIVE pabs are specific for the dually phosphorylated active forms of MAPK, p38 and JNK, and provide a tool for more accurate measurement of activation of these enzymes. Anti-ACTIVE MAPK pab, Rabbit (Cat.# V8031), is an affinity-purified polyclonal antibody that specifically recognizes the dually phosphorylated, active form of MAPK (also known as p44/erk1 and p42/erk2) enzymes. Anti-ACTIVE MAPK pab is raised against a dually phosphorylated peptide sequence representing the catalytic core of the active ERK enzyme. The phosphorylated amino acid residues correspond to Thr 183 and Tyr 185 of the p42/erk2 enzyme. The recommended dilution of Anti-ACTIVE MAPK pab for Western blot analysis is 1:5,000. Revised 1/12 Page 1

3 1. Description (continued) Anti-ACTIVE JNK pab, Rabbit (Cat.# V7931, V7932), is an affinity-purified polyclonal antibody that recognizes the dually phosphorylated, active form of JNK (c-jun N-terminal protein kinase), also known as SAPK (Stress-Activated Protein Kinase). Anti-ACTIVE JNK pab is raised against a dually phosphorylated peptide sequence representing the catalytic core of the active JNK enzyme. The phosphorylated amino acid residues correspond to Thr 183 and Tyr 185 of the JNK2 enzyme. The recommended dilution of Anti-ACTIVE JNK pab for Western blot analysis is 1:5,000. Anti-ACTIVE p38 pab, Rabbit (Cat.# V1211), is an affinity-purified polyclonal antibody that recognizes the active form of p38 enzyme. The Anti-ACTIVE p38 pab is raised against the dually phosphorylated peptide sequence representing the catalytic core of the active p38 enzyme. The phosphorylated amino acid residues correspond to Thr 180 and Tyr 182 of the p38 enzyme. The recommended dilution of Anti-ACTIVE p38 pab for Western blot analysis is 1:2,000. Donkey Anti-Rabbit IgG (H+L), HRP is an affinity-purified horseradish peroxidase (HRP)-conjugated secondary antibody for use with the Anti-ACTIVE pabs. It is qualified for use in Western blot analysis using chemiluminescent and colorimetric detection methods. The antibody conjugate exhibits minimal cross-reactivity to goat, mouse and sheep IgG, bovine serum albumin (BSA) and proteins in mammalian cell extracts. The HRP-antibody conjugate provides low backgrounds and highly specific signals when used at the recommended 1:5,000 dilution with Anti-ACTIVE MAPK pab, and 1:10,000 dilution with Anti-ACTIVE JNK and Anti-ACTIVE p38 pabs. Page 2 Revised 1/12

4 2. Product Components and Storage Conditions Product Size Cat.# Anti ACTIVE MAPK pab, Rabbit (ptepy) 40µl V8031 Anti-ACTIVE JNK pab, Rabbit (ptppy) 40µl V µl V7932 Anti-ACTIVE p38 pab, Rabbit (ptgpy) 100µl V1211 Donkey Anti-Rabbit IgG (H+L), HRP, Anti-ACTIVE Qualified* 60µl V7951 *For Laboratory Use. When used at the recommended dilutions, Cat.# V7932 and V7951 will generate 600ml of blotting solution, and Cat.# V1211, V8031 and V7931 will generate 200ml of blotting solution. Each system includes: 1 tube Anti-ACTIVE pab or Anti-ACTIVE Qualified Antibody Conjugate Storage Conditions: Store the antibodies and conjugate at 20 C, where they are stable for six months from the date of purchase unless otherwise stated on the label. To avoid multiple freeze-thaw cycles, we recommend dispensing the antibodies into aliquots prior to storage. 3. General Considerations Mitogen-Activated Protein Kinases (MAPKs) play an important role in signal transduction in eukaryotic cells where they modulate many cellular events (1,2). The MAP Kinase superfamily includes the ERKs, JNKs and p38 kinases that are found in three interwoven signal transduction cascades. These kinases phosphorylate, and thus activate, transcription factors in response to mitogens, growth factors or various forms of stress. ERK, JNK and p38, when activated by MAP Kinase kinases, known as MEKs, undergo phosphorylation on the threonine and tyrosine residues in the sequence ptxpy. Thus, both threonine and tyrosine phosphorylation are necessary for maximal activation of ERK, JNK and p38. Revised 1/12 Page 3

5 3. General Considerations (continued) The ERK, or Extracellular signal-regulated protein Kinase family is composed of five reported isoforms, ERK1 through ERK5. JNK, or c-jun N-terminal Kinase, phosphorylates the transcription factor c-jun (AP1). JNK is also known as SAPK. Major isoforms of JNK are p46 JNK1, p54 JNK2, and p49 JNK3 (3), although there can be up to 10 splice variants (4). In the case of p38, the protein was originally called CSBP for Cytokine-Suppressive anti-inflammatory drug Binding Protein. A yeast p38 homolog, HOG (High Osmolarity Glycerol response), has been characterized. The p38 protein phosphorylates the Activating Transcription Factor (ATF) family. The p38 isoforms are designated p38, p38b, p38g and p38d and show slight differences in mobility during SDS-PAGE (5). The JNK/SAPK and p38/hog pathways are activated by ultraviolet light, cytokines, osmotic shock agents such as sorbitol, inhibitors of protein synthesis such as anisomycin, and to a lesser extent by growth factors. This spectrum of regulators suggests that the enzymes are transducers of a variety of stress responses. These signals are transmitted from cell membrane-bound receptors through a variety of small GTP-binding proteins, such as Cdc42 or Rac in the case of JNK and p38 (6), to the level of the MEK Kinases (MEKKs), then to the MEK enzymes. JNK and p38 then are activated by their respective kinases, MEK4/7 and MEK3/6, but MEK activation depends upon both stimulus and cell type (7). For example, a large, transient rise in calcium ion concentration in B cells can activate MEK4/7 and JNK. A lower sustained level of Ca 2+ does not (8). JNK and p38 homologs are found in organisms at many different phylogenetic levels. In Drosophila, the basket (bsk) gene encodes a JNK homolog essential for successful embryonic development (9) and immune responsiveness (10). In yeast, the p38/hog homolog pathway can be activated by osmotic shock and is regulated by a MEK called Pbs2p (11). Page 4 Revised 1/12

6 A. Anti-ACTIVE MAPK NGF kda + Anti-ACTIVE JNK Sorbitol + Anti-ACTIVE p38 Sorbitol JNK2 ERK1 ERK2 JNK1 p B. Anti-MAPK NGF kda + Anti-JNK Sorbitol + Anti-p38 Sorbitol JNK2 ERK1 JNK1 ERK2 p TC Figure 1. Detection of activated MAPK, JNK and p38 in PC12 cell extracts by Western blot analysis using Anti-ACTIVE MAPK, JNK and p38 Polyclonal Antibodies. PC12 cells were grown to 60 80% confluence in RPMI 1640 medium supplemented with 25mM HEPES, 0.5mM EGTA, 10% horse serum and 5% fetal bovine serum. Cells were either untreated, treated with 50ng/ml of nerve growth factor (NGF) for 5 minutes, or 0.5M sorbitol for 5 minutes, as indicated. Cells were harvested, homogenized and subjected to high-speed centrifugation (7). The resulting supernatants were stored at 70 C. Aliquots of each extract were analyzed by SDS-PAGE (10% gel, under reducing conditions) and transferred to nitrocellulose membranes. The membranes were probed with the indicated Anti-ACTIVE pab (Panel A) or with the corresponding antibodies that recognize both active and inactive forms of each subfamily of kinases (Panel B). For JNK, the antibody that detects both forms was generated against whole JNK enzyme from rat, while the corresponding antibodies for p38 and ERK1 and ERK2 were generated against synthetic peptides derived from the deduced amino acid sequence of each protein. The secondary antibody was anti-rabbit IgG (H + L). Chemiluminescent detection was performed using an Applera Corporation Western-Star Kit and Kodak BioMax film, as described by the manufacturer. Lanes 1, 5 and 9, 2µg of unstimulated PC12 cell extract; lanes 2, 6 and 10, 2µg of NGF-stimulated PC12 cell extract; lanes 3, 7 and 11, 20µg of unstimulated PC12 cell extract; lanes 4, 8 and 12, 20µg of sorbitol-treated PC12 cell extract. Revised 1/12 Page 5

7 3. General Considerations (continued) A. Nitrocellulose B. PVDF Perform SDS-PAGE and transfer to a nitrocellulose membrane. Perform SDS-PAGE and transfer to a PVDF membrane. Block nitrocellulose membrane with TBS/1% BSA for 1 hour (37 C) or overnight (4 C). Block PVDF membrane with PVDF Buffer for 1 hour (37 C) or overnight (4 C). Apply Anti-ACTIVE pab diluted with TBST/0.1% BSA and incubate 2 hours at room temperature with agitation. Apply Anti-ACTIVE pab diluted with PVDF Buffer and incubate 2 hours at room temperature with agitation. Wash membrane 3 times with 75ml of TBST (15 minutes each); decant after each wash. Wash membrane 3 times with 75ml of PVDF Buffer (15 minutes each); decant after each wash. Dilute Anti-Rabbit Antibody conjugate with TBST/0.1% BSA*. Incubate 1 hour at room temperature with agitation. Dilute Anti-Rabbit Antibody conjugate with PVDF Buffer*. Incubate 1 hour at room temperature with agitation. Wash membrane 3 times (15 minutes each) in 75ml of TBST. Rinse membrane twice (1 minute each) in TBS; decant after each wash. Wash membrane 3 times (15 minutes each) in 75ml of PVDF Buffer. Rinse membrane twice (1 minute each) in TBS; decant after each wash. Colorimetric Detection Incubate with detection reagent until appropriate signal level is obtained. HRP: KPL TMB Reagent. Chemiluminescent Detection HRP: Soak blot for 1 minute in ECL Detection Reagent. Expose blot to film. Colorimetric Detection Incubate with detection reagent until appropriate signal level is obtained. HRP: KPL TMB Reagent. Chemiluminescent Detection HRP: Soak blot for 1 minute in ECL Detection Reagent. Expose blot to film. 1851MA01_8A Figure 2. Schematic diagram illustrating the use of nitrocellulose and PVDF membranes in Western blot analysis with Anti-ACTIVE pabs. Protocols for use with nitrocellulose (Panel A) and PVDF (Panel B) membranes. Recommended dilutions of the Anti-ACTIVE pabs are 1:5,000 for Anti-ACTIVE MAPK pab, 1:2,000 for Anti-ACTIVE p38 pab, 1:5,000 for Anti-ACTIVE JNK pab. Dilute Anti- ACTIVE Qualified Donkey Anti-Rabbit IgG (H + L), HRP-conjugated secondary antibody 1:5,000 to 1:10,000. *Dilute anti-rabbit IgG (H + L) AP-conjugated secondary antibody as instructed by the manufacturer. KPL is an abbreviation for Kirkegaard and Perry Laboratories. Note: It may be necessary to empirically determine the optimal dilutions of primary and secondary antibodies for your experimental system. Use of secondary antibodies other than those available from Promega may require additional optimization. Page 6 Revised 1/12

8 The Anti-ACTIVE antibodies are directed against synthetic peptides encompassing residues ptepy of ERK2 (12), the ptgpy motif of p38a or the ptppy motif of JNK2 (13). All three antibodies are affinity-purified by a twostep procedure. The antibodies are first purified by immunodepletion with a nonphosphorylated peptide, followed by positive immunoaffinity selection with a dually phosphorylated peptide derived from the active enzyme. Thus, the final antibody preparation is specific to the active, ptxpy sequence of the respective kinase. Figure 1 demonstrates results obtained using the Anti-ACTIVE MAPK, JNK and p38 pabs in Western blot analyses. 4. Western Blot Analysis with Anti-ACTIVE pabs Figure 2 outlines the procedure for the use of Anti-ACTIVE pabs in Western blot analysis. This procedure results in strong signal and very low backgrounds with a variety of experimental systems; however, some optimization may be required for best results with your particular application. Materials to Be Supplied by the User (Solution compositions are provided in Section 6.) TMB reagent for horseradish peroxidase (HRP) colorimetric detection (KPL Cat.# ) Western Blue Stabilized Substrate for alkaline phosphatase (AP) colorimetric detection (Cat.# S3841) Western-Star Substrate for chemiluminescent detection (Applera Corporation Cat.# WL10RS) ECL detection reagent for chemiluminescent detection (Amersham Biosciences Cat.# RPN 2109) secondary antibodies [either Donkey Anti-Rabbit IgG (H+L), HRP, Anti- ACTIVE Qualified (Cat.# V7951) or anti-rabbit IgG (H + L), AP] Blot Qualified BSA (Cat.# W3841) TBST and TBS buffers (for nitrocellulose membranes) PVDF buffer (for PVDF membranes) 4.A.! Probing Membranes with Anti-ACTIVE pabs 1. Perform SDS-PAGE analysis, then transfer to either a nitrocellulose or PVDF membrane. 2. Block the nitrocellulose membrane with TBS buffer/1% BSA for 1 hour at 37 C or overnight at 4 C. Block the PVDF membrane with PVDF buffer for 1 hour at 37 C or overnight at 4 C. Do not use dried milk to block membranes. Revised 1/12 Page 7

9 4.A. Probing Membranes with Anti-ACTIVE pabs (continued) Note: It may be necessary to empirically determine the optimal dilutions of primary and secondary antibodies for your experimental system. 3. Prepare the primary antibody for incubation with the membrane. Recommended dilutions for the Anti-ACTIVE pabs are 1:5,000 for Anti-ACTIVE MAPK (Cat.# V8031), 1:5,000 for Anti-ACTIVE JNK (Cat.# V7931 and V7932) and 1:2,000 for Anti-ACTIVE p38 (Cat.# V1211). Nitrocellulose membranes: Add the Anti-ACTIVE pab, diluted with TBST/0.1% BSA, and incubate for 2 hours at room temperature with agitation. PVDF membranes: Dilute the Anti-ACTIVE pab with PVDF buffer, then incubate as above. 4. Nitrocellulose membranes: Wash 3 times with 75ml of TBST buffer, 15 minutes per wash. Decant and replace the buffer after each wash. PVDF membranes: Wash the PVDF membrane 3 times, as above, using 75ml of PVDF buffer for each wash. Decant and replace the buffer after each wash. 5. Prepare the secondary antibody conjugate for incubation with the membrane. The recommended dilution for the Anti-ACTIVE Qualified Donkey Anti-Rabbit antibody conjugate, HRP, is 1:5,000 to 1:10,000. If using an anti-rabbit IgG (H + L) AP-conjugated secondary antibody, dilute as instructed by the manufacturer. Nitrocellulose membranes: Dilute the Donkey Anti-Rabbit Antibody conjugate (1:5,000 to 1:10,000) with TBST/0.1% BSA. Incubate for 1 hour at room temperature with agitation. PVDF membranes: Dilute the Donkey Anti-Rabbit conjugate (1:5,000 to 1:10,000) with PVDF buffer and incubate as above. 6. Nitrocellulose membranes: Wash the membrane 3 times, 15 minutes each wash, in 75ml TBST buffer. Rinse the membrane 2 times, for 1 minute each, in TBS buffer. Decant the solution after each wash and after each rinse. PVDF membranes: Wash 3 times as above, using PVDF buffer. Rinse 2 times, 1 minute each, in TBS buffer. Decant after each wash and after each rinse. 4.B. Detection Protocols Two types of detection can be used to analyze gel results on nitrocellulose or PVDF membranes. Select either colorimetric or chemiluminescent detection, and follow the appropriate instructions below. Colorimetric Detection: For both nitrocellulose and PVDF membranes, incubate with the detection reagent until the appropriate signal level is obtained. Page 8 Revised 1/12

10 Chemiluminescent Detection using Horseradish Peroxidase: For both nitrocellulose and PVDF membranes, soak the blot for 1 minute in ECL Detection Reagent. Expose the blot to film. Chemiluminescent Detection using Alkaline Phosphatase: Soak the nitrocellulose or PVDF blot for 5 minutes in Western-Star Substrate (Applera Corporation). Remove the excess reagent, and expose the blot to film. 5. Immunocytochemistry Protocol for Anti-ACTIVE pabs Materials to Be Supplied by the User (Solution compositions are provided in Section 6.) Lab-Tek 4-chambered slides (Fisher Cat.# ) rat tail collagen (Collaborative BioScience Products) RPMI 1640 with 25mM HEPES, 300mg/L L-glutamine, 10% horse serum, 5% fetal bovine serum and 0.5mM EGTA NGF (Cat.# G5141) or sorbitol PBS 10% paraformaldehyde methanol ( 20 C) blocking buffer (1% BSA, 5% donkey serum in PBS) antibody dilution buffer (1% BSA, 1% donkey serum in PBS) Donkey Anti-Rabbit Cy 3 conjugate (Jackson ImmunoResearch Cat.# ) The following method is for preparing and immunostaining PC12 cells stimulated by either nerve growth factor (NGF) to activate MAP kinases or sorbitol to activate JNK and p38 kinases. Figure 3 demonstrates results obtained using the Anti-ACTIVE pabs for immunocytochemical (ICC) staining of PC12 cells. 1. Coat Lab-Tek 4-chambered slides with rat tail collagen (6µg/cm 2 in sterile PBS) for 1 hour. 2. Grow PC12 cells in chambers at 37 C in 5% CO 2 in medium containing RPMI 1640 with 25mM HEPES, 300mg/L L-glutamine, 10% horse serum, 5% fetal bovine serum and 0.5mM EGTA. The medium should be changed every other day until cells reach 80% confluence. 3. Activate the cells in two chambers as described below. Use the cells in the remaining two chambers as untreated controls. NGF Treatment to Activate ERK The day before immunocytochemistry (ICC), add fresh medium with serum. The next day, add 200ng/ml NGF in RPMI. Incubate for 5 minutes at 37 C. Revised 1/12 Page 9

11 5. Immunocytochemistry Protocol for Anti-ACTIVE pabs (continued) Sorbitol Treatment to Activate JNK and p38 Kinase The day before ICC, add fresh medium without serum. The next day, add sorbitol to a final concentration of 1M. Incubate for 30 minutes at 37 C. 4. Wash once with cold PBS. 5. Fix cells with 10% paraformaldehyde for 30 minutes at room temperature. 6. Wash for 5 minutes with PBS. Repeat twice for a total of 3 washes. 7. Permeabilize the cells with cold ( 20 C) methanol for 10 minutes. 8. Wash for 5 minutes with PBS. Repeat twice for a total of 3 washes. 9. Incubate with blocking buffer (1% BSA, 5% donkey serum in PBS) for 3 hours at room temperature. 10. Wash once for 5 minutes with PBS. Note: It may be necessary to empirically determine the optimal dilutions of primary and secondary antibodies for your experimental system. 11. Incubate with Anti-ACTIVE pab in antibody dilution buffer (1% BSA, 1% donkey serum in PBS) overnight at 4 C. Recommended antibody dilutions are: MAPK 1:500; JNK 1:1,000; p38 1: Wash for 15 minutes with PBS. Repeat four times for a total of 5 washes. 13. Incubate for 90 minutes at room temperature with Donkey Anti-Rabbit Cy 3 Conjugate at a 1:1,000 dilution in antibody dilution buffer (1% BSA, 1% donkey serum in PBS). 14. Wash for 15 minutes with PBS. Repeat four times for a total of 5 washes. 15. Remove grid, and mount the slides with Vectashield containing DAPI. Page 10 Revised 1/12

12 tb262v13final:eivd_tm.qxd 2/2/2012 1:24 PM Page 11 A. Anti-ACTIVE MAPK pab NGF-Treated Untreated B. Anti-ACTIVE JNK pab Sorbitol-Treated Untreated C. Anti-ACTIVE p38 pab Untreated 2870TA02_0A Sorbitol-Treated Figure 3. Detection of activated MAPK, JNK and p38 in PC12 cells by immunocytochemistry (ICC). PC12 cells were grown to 80% confluence in RPMI 1640 medium supplemented with 25mM HEPES, 300mg/L L-glutamine, 10% horse serum, 5% fetal bovine serum and 0.5mM EGTA. Cells were either untreated or treated with 200ng/ml NGF or 1M sorbitol as indicated. ICC was performed as described (Section 5). Anti-ACTIVE antibodies were used at the following dilutions: MAPK, 1:500 (Panel A); JNK, 1:1,000 (Panel B); p38, 1:500 (Panel C). Revised 1/12 Page 11

13 6. Composition of Buffers and Solutions 10% paraformaldehyde 5g paraformaldehyde) 50ml PBS Heat at 75 C with stirring in a fume hood until dissolved. Cool to room temperature. Adjust ph to 7.2 with 10N NaOH. Add deionized water to a final volume of 50ml. PVDF buffer TBS buffer with 0.2% I-Block (Applera Corporation) and 0.1% Tween 20 TBS buffer 20mM Tris-HCl (ph 7.5) 150mM NaCl TBST buffer TBS buffer with 0.05% Tween References 1. Seger, R. and Krebs, E.G. (1995) The MAPK signaling cascade. FASEB J. 9, Cano, E. and Mahadevan, L.C. (1995) Parallel signal processing among mammalian MAPKs. Trends Biochem. Sci. 20, Kyriakis, J.M. et al. (1994) The stress-activated protein kinase subfamily of c-jun kinases. Nature 369, Gupta, S. et al. (1996) Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15, Li, Z. et al. (1996) The primary structure of p38 gamma: A new member of p38 group of MAP kinases. Biochem. Biophys. Res. Comm. 228, Lamarche, N. et al. (1996) Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65 PAK and the JNK/SAPK MAP kinase cascade. Cell 87, Meier, R. et al. (1996) Cellular stresses and cytokines activate multiple mitogenactivated-protein kinase kinase homologues in PC12 and KB cells. Eur. J. Biochem. 236, Dolmetsch, R.E. et al. (1997) Differential activation of transcription factors induced by Ca 2+ response amplitude and duration. Nature 386, Riesgo-Escovar, J.R. et al. (1996) The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye. Genes Dev. 10, Sluss, H.K. et al. (1996) A JNK signal transduction pathway that mediates morphogenesis and an immune response in Drosophila. Genes Dev. 10, Posas, F. and Saito, H. (1997) Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: Scaffold role of Pbs2p MAPKK. Science 276, Payne, D.M. et al. (1991) Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 10, Derijard, B. et al. (1994) JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-jun activation domain. Cell 76, Page 12 Revised 1/12

14 8. Related Products Product Size Cat. # Anti-ERK 1/2 pab, Rabbit 40µl V1141 Anti-pT 183 MAPK pab, Rabbit 50µl V8081 MEK Inhibitor U0126 5mg V1121 PD mg V1191 SB mg V1161 mngf, 2.5S 100µg G µg G Promega Corporation. All Rights Reserved. Anti-ACTIVE and Western Blue are registered trademarks of Promega Corporation. BioMax and Kodak are registered trademarks of Eastman Kodak. ECL is a trademark of and Cy is a registered trademark of Amersham Biosciences. Lab-Tek is a registered trademark of Nalge-Nunc International. Tween is a registered trademark of ICI Americas, Inc. I-Block and Western-Star are trademarks of Applera Corporation. Vectashield is a registered trademark of Vector Laboratories, Inc. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. Revised 1/12 Page 13