mapk, p38 and jnk polyclonal antibodies - promega

T e c h n i c a l B u l l e t i n

Anti-ACTIVE® MAPK, p38and JNK PolyclonalAntibodies and Anti-ACTIVE® QualifiedSecondary AntibodyConjugateINSTRUCTIONS FOR USE OF PRODUCTS V1211, V8031, V7931, V7932AND V7951.

PRINTED IN USA.Revised 1/12. Part# TB262

tb262v13final:EIVD_TM.qxd 2/2/2012 1:24 PM

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TB262Revised 1/12 Page 1

1. Description ..........................................................................................................1

2. Product Components and Storage Conditions ............................................3

3. General Considerations ....................................................................................3

4. Western Blot Analysis with Anti-ACTIVE® pAbs......................................7A. Probing Membranes with Anti-ACTIVE® pAbs..............................................7B. Detection Protocols ..............................................................................................8

5. Immunocytochemistry Protocol for Anti-ACTIVE® pAbs ........................9

6. Composition of Buffers and Solutions .......................................................12

7. References .........................................................................................................12

8. Related Products ..............................................................................................13

1. Description

Promega has developed polyclonal antibodies (pAb) to three members of theMitogen-Activated Protein Kinase (MAPK) superfamily of enzymes. Theseenzymes, which play important roles in signal transduction in eukaryotic cells,are MAPK, p38 and JNK. These kinases have interconnected roles in a cascadeof signal transduction reactions. The Anti-ACTIVE® pAbs are specific for thedually phosphorylated active forms of MAPK, p38 and JNK, and provide a toolfor more accurate measurement of activation of these enzymes.

Anti-ACTIVE® MAPK pAb, Rabbit (Cat.# V8031), is an affinity-purifiedpolyclonal 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 peptidesequence representing the catalytic core of the active ERK enzyme. Thephosphorylated amino acid residues correspond to Thr 183 and Tyr 185 of thep42/ERK2 enzyme. The recommended dilution of Anti-ACTIVE® MAPK pAbfor Western blot analysis is 1:5,000.

Anti-ACTIVE® MAPK, p38 andJNK Polyclonal Antibodies andAnti-ACTIVE® QualifiedSecondary Antibody ConjugateAll technical literature is available on the Internet at www.promega.com/resources/protocols/

Please visit the web site to verify that you are using the most current version of thisTechnical Bulletin. Please contact Promega Technical Services if you have questions on use

of this system. E-mail [email protected].

tb262v13final:EIVD_TM.qxd 2/2/2012 1:24 PM Page 1

1. Description (continued)

Anti-ACTIVE® JNK pAb, Rabbit (Cat.# V7931, V7932), is an affinity-purifiedpolyclonal antibody that recognizes the dually phosphorylated, active form of JNK(c-Jun N-terminal protein kinase), also known as SAPK (Stress-Activated ProteinKinase). Anti-ACTIVE® JNK pAb is raised against a dually phosphorylatedpeptide sequence representing the catalytic core of the active JNK enzyme. Thephosphorylated amino acid residues correspond to Thr 183 and Tyr 185 of theJNK2 enzyme. The recommended dilution of Anti-ACTIVE® JNK pAb for Westernblot analysis is 1:5,000.

Anti-ACTIVE® p38 pAb, Rabbit (Cat.# V1211), is an affinity-purified polyclonalantibody that recognizes the active form of p38 enzyme. The Anti-ACTIVE® p38pAb is raised against the dually phosphorylated peptide sequence representingthe catalytic core of the active p38 enzyme. The phosphorylated amino acidresidues correspond to Thr 180 and Tyr 182 of the p38 enzyme. The recommendeddilution of Anti-ACTIVE® p38 pAb for Western blot analysis is 1:2,000.

Donkey Anti-Rabbit IgG (H+L), HRP is an affinity-purified horseradishperoxidase (HRP)-conjugated secondary antibody for use with the Anti-ACTIVE® pAbs. It is qualified for use in Western blot analysis usingchemiluminescent and colorimetric detection methods. The antibody conjugateexhibits minimal cross-reactivity to goat, mouse and sheep IgG, bovine serumalbumin (BSA) and proteins in mammalian cell extracts. The HRP-antibodyconjugate provides low backgrounds and highly specific signals when used atthe recommended 1:5,000 dilution with Anti-ACTIVE® MAPK pAb, and 1:10,000dilution with Anti-ACTIVE® JNK and Anti-ACTIVE® p38 pAbs.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TB262 Printed in USA.Page 2 Revised 1/12


2. Product Components and Storage Conditions

Product Size Cat.#Anti ACTIVE® MAPK pAb, Rabbit (pTEpY) 40µl V8031Anti-ACTIVE® JNK pAb, Rabbit (pTPpY) 40µl V7931

120µl V7932Anti-ACTIVE® p38 pAb, Rabbit (pTGpY) 100µl V1211Donkey 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 ofblotting 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 arestable for six months from the date of purchase unless otherwise stated on the label.To avoid multiple freeze-thaw cycles, we recommend dispensing the antibodiesinto aliquots prior to storage.

3. General Considerations

Mitogen-Activated Protein Kinases (MAPKs) play an important role in signaltransduction in eukaryotic cells where they modulate many cellular events (1,2).The MAP Kinase superfamily includes the ERKs, JNKs and p38 kinases that arefound in three interwoven signal transduction cascades. These kinasesphosphorylate, and thus activate, transcription factors in response to mitogens,growth factors or various forms of stress. ERK, JNK and p38, when activated byMAP Kinase kinases, known as MEKs, undergo phosphorylation on thethreonine and tyrosine residues in the sequence pTXpY. Thus, both threonineand tyrosine phosphorylation are necessary for maximal activation of ERK, JNKand p38.



3. General Considerations (continued)

The ERK, or Extracellular signal-Regulated protein Kinase family is composedof five reported isoforms, ERK1 through ERK5. JNK, or c-Jun N-terminalKinase, phosphorylates the transcription factor c-Jun (AP1). JNK is also knownas 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 proteinwas originally called CSBP for Cytokine-Suppressive anti-inflammatory drugBinding Protein. A yeast p38 homolog, HOG (High Osmolarity Glycerolresponse), has been characterized. The p38 protein phosphorylates theActivating Transcription Factor (ATF) family. The p38 isoforms are designatedp38, 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 synthesissuch as anisomycin, and to a lesser extent by growth factors. This spectrum ofregulators suggests that the enzymes are transducers of a variety of stressresponses. These signals are transmitted from cell membrane-bound receptorsthrough a variety of small GTP-binding proteins, such as Cdc42 or Rac in thecase of JNK and p38 (6), to the level of the MEK Kinases (MEKKs), then to theMEK enzymes. JNK and p38 then are activated by their respective kinases,MEK4/7 and MEK3/6, but MEK activation depends upon both stimulus andcell type (7). For example, a large, transient rise in calcium ion concentration inB cells can activate MEK4/7 and JNK. A lower sustained level of Ca2+ does not(8). JNK and p38 homologs are found in organisms at many differentphylogenetic levels. In Drosophila, the basket (bsk) gene encodes a JNK homologessential for successful embryonic development (9) and immuneresponsiveness (10). In yeast, the p38/HOG homolog pathway can be activatedby osmotic shock and is regulated by a MEK called Pbs2p (11).




Figure 1. Detection of activated MAPK, JNK and p38 in PC12 cell extracts byWestern blot analysis using Anti-ACTIVE® MAPK, JNK and p38 PolyclonalAntibodies. PC12 cells were grown to 60–80% confluence in RPMI 1640 mediumsupplemented with 25mM HEPES, 0.5mM EGTA, 10% horse serum and 5% fetalbovine serum. Cells were either untreated, treated with 50ng/ml of nerve growthfactor (NGF) for 5 minutes, or 0.5M sorbitol for 5 minutes, as indicated. Cells wereharvested, homogenized and subjected to high-speed centrifugation (7). Theresulting supernatants were stored at –70°C. Aliquots of each extract were analyzedby SDS-PAGE (10% gel, under reducing conditions) and transferred to nitrocellulosemembranes. The membranes were probed with the indicated Anti-ACTIVE® pAb(Panel A) or with the corresponding antibodies that recognize both active andinactive forms of each subfamily of kinases (Panel B). For JNK, the antibody thatdetects both forms was generated against whole JNK enzyme from rat, while thecorresponding antibodies for p38 and ERK1 and ERK2 were generated againstsynthetic peptides derived from the deduced amino acid sequence of each protein.The secondary antibody was anti-rabbit IgG (H + L). Chemiluminescent detectionwas performed using an Applera Corporation Western-Star™ Kit and Kodak®

BioMax® film, as described by the manufacturer. Lanes 1, 5 and 9, 2µg ofunstimulated PC12 cell extract; lanes 2, 6 and 10, 2µg of NGF-stimulated PC12 cellextract; lanes 3, 7 and 11, 20µg of unstimulated PC12 cell extract; lanes 4, 8 and 12,20µg of sorbitol-treated PC12 cell extract.

1859

TC

1 2 3 4 5 6

– ERK1– JNK2– JNK1

– ERK2 – p38

98 –

64 –

50 –

36 –

kDa – + NGF

Anti-ACTIVE® MAPK

– + Sorbitol

Anti-ACTIVE® JNK

– + Sorbitol

Anti-ACTIVE® p38

1 2 3 4 5 6

– ERK1

– JNK2

– JNK1– ERK2 – p38

98 –

64 –

50 –

36 –

kDa – + NGFAnti-MAPK

– + SorbitolAnti-JNK

– + SorbitolAnti-p38

A.

B.



3. General Considerations (continued)

Figure 2. Schematic diagram illustrating the use of nitrocellulose and PVDFmembranes in Western blot analysis with Anti-ACTIVE® pAbs. Protocols for usewith nitrocellulose (Panel A) and PVDF (Panel B) membranes. Recommendeddilutions 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 secondaryantibody 1:5,000 to 1:10,000. *Dilute anti-rabbit IgG (H + L) AP-conjugatedsecondary antibody as instructed by the manufacturer. KPL is an abbreviation forKirkegaard and Perry Laboratories.Note: It may be necessary to empirically determine the optimal dilutions of primaryand secondary antibodies for your experimental system. Use of secondary antibodiesother than those available from Promega may require additional optimization.

1851MA01_8A

Perform SDS-PAGE and transfer to a PVDF membrane.

Block PVDF membrane with PVDF Buffer for 1 hour (37°C) or overnight (4°C).

Apply Anti-ACTIVE® pAb diluted with PVDF Buffer and incubate 2 hours at

room temperature with agitation.

Wash membrane 3 times with 75ml of PVDF Buffer (15 minutes each);

decant after each wash.

Dilute Anti-Rabbit Antibody conjugate with PVDF Buffer*.

Incubate 1 hour at roomtemperature with agitation.

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 DetectionIncubate with detection reagent until appropriate signal level is obtained.HRP: KPL TMB Reagent.

Chemiluminescent DetectionHRP: Soak blot for 1 minute in ECL™ Detection Reagent.Expose blot to film.

B. PVDF

Perform SDS-PAGE and transfer to a nitrocellulose membrane.

Block nitrocellulose membrane with TBS/1% BSA 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.

Wash membrane 3 times with 75ml of TBST (15 minutes each); decant

after each wash.

Dilute Anti-Rabbit Antibody conjugate with TBST/0.1% BSA*.

Incubate 1 hour at roomtemperature 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.

Colorimetric DetectionIncubate with detection reagent until appropriate signal level is obtained.HRP: KPL TMB Reagent.

Chemiluminescent DetectionHRP: Soak blot for 1 minute in ECL™ Detection Reagent.Expose blot to film.

A. Nitrocellulose



The Anti-ACTIVE® antibodies are directed against synthetic peptidesencompassing residues pTEpY of ERK2 (12), the pTGpY motif of p38a or thepTPpY motif of JNK2 (13). All three antibodies are affinity-purified by a two-step procedure. The antibodies are first purified by immunodepletion with anonphosphorylated peptide, followed by positive immunoaffinity selectionwith a dually phosphorylated peptide derived from the active enzyme. Thus,the final antibody preparation is specific to the active, pTXpY sequence of therespective 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 blotanalysis. This procedure results in strong signal and very low backgrounds with avariety of experimental systems; however, some optimization may be required forbest 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.# 50-77-00)• 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 orPVDF membrane.

2. Block the nitrocellulose membrane with TBS buffer/1% BSA for 1 hour at37°C or overnight at 4°C. Block the PVDF membrane with PVDF buffer for 1 hour at 37°C orovernight at 4°C.

Do not use dried milk to block membranes.!



4.A. Probing Membranes with Anti-ACTIVE® pAbs (continued)

Note: It may be necessary to empirically determine the optimal dilutions ofprimary 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 withTBST/0.1% BSA, and incubate for 2 hours at room temperature withagitation.

PVDF membranes: Dilute the Anti-ACTIVE® pAb with PVDF buffer, thenincubate 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, using75ml of PVDF buffer for each wash. Decant and replace the buffer aftereach wash.

5. Prepare the secondary antibody conjugate for incubation with themembrane. The recommended dilution for the Anti-ACTIVE® QualifiedDonkey Anti-Rabbit antibody conjugate, HRP, is 1:5,000 to 1:10,000. If usingan anti-rabbit IgG (H + L) AP-conjugated secondary antibody, dilute asinstructed by the manufacturer.

Nitrocellulose membranes: Dilute the Donkey Anti-Rabbit Antibodyconjugate (1:5,000 to 1:10,000) with TBST/0.1% BSA. Incubate for 1 hour atroom temperature with agitation.

PVDF membranes: Dilute the Donkey Anti-Rabbit conjugate (1:5,000 to1:10,000) with PVDF buffer and incubate as above.

6. Nitrocellulose membranes: Wash the membrane 3 times, 15 minutes eachwash, 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. Rinse2 times, 1 minute each, in TBS buffer. Decant after each wash and after eachrinse.

4.B. Detection Protocols

Two types of detection can be used to analyze gel results on nitrocellulose orPVDF 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 isobtained.


Chemiluminescent Detection using Horseradish Peroxidase: For bothnitrocellulose and PVDF membranes, soak the blot for 1 minute in ECL™Detection Reagent. Expose the blot to film.

Chemiluminescent Detection using Alkaline Phosphatase: Soak thenitrocellulose or PVDF blot for 5 minutes in Western-Star™ Substrate (AppleraCorporation). 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.# 12-565-21)• 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.# 741-165-152)

The following method is for preparing and immunostaining PC12 cells stimulatedby either nerve growth factor (NGF) to activate MAP kinases or sorbitol toactivate JNK and p38 kinases. Figure 3 demonstrates results obtained using theAnti-ACTIVE® pAbs for immunocytochemical (ICC) staining of PC12 cells.

1. Coat Lab-Tek® 4-chambered slides with rat tail collagen (6µg/cm2 in sterilePBS) for 1 hour.

2. Grow PC12 cells in chambers at 37°C in 5% CO2 in medium containingRPMI 1640 with 25mM HEPES, 300mg/L L-glutamine, 10% horse serum,5% fetal bovine serum and 0.5mM EGTA. The medium should be changedevery other day until cells reach 80% confluence.

3. Activate the cells in two chambers as described below. Use the cells in theremaining two chambers as untreated controls.

NGF Treatment to Activate ERKThe day before immunocytochemistry (ICC), add fresh medium withserum. The next day, add 200ng/ml NGF in RPMI. Incubate for 5 minutesat 37°C.



5. Immunocytochemistry Protocol for Anti-ACTIVE® pAbs (continued)

Sorbitol Treatment to Activate JNK and p38 KinaseThe day before ICC, add fresh medium without serum. The next day, addsorbitol 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) for3 hours at room temperature.

10. Wash once for 5 minutes with PBS.Note: It may be necessary to empirically determine the optimal dilutions ofprimary 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 dilutionsare: MAPK 1:500; JNK 1:1,000; p38 1:500.

12. 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-RabbitCy®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.



Figure 3. Detection of activated MAPK, JNK and p38 in PC12 cells byimmunocytochemistry (ICC). PC12 cells were grown to 80% confluence in RPMI1640 medium supplemented with 25mM HEPES, 300mg/L L-glutamine, 10% horseserum, 5% fetal bovine serum and 0.5mM EGTA. Cells were either untreated ortreated with 200ng/ml NGF or 1M sorbitol as indicated. ICC was performed asdescribed (Section 5). Anti-ACTIVE® antibodies were used at the followingdilutions: MAPK, 1:500 (Panel A); JNK, 1:1,000 (Panel B); p38, 1:500 (Panel C).


2870TA02_0A

A. Anti-ACTIVE® MAPK pAbNGF-Treated Untreated

B. Anti-ACTIVE® JNK pAbSorbitol-Treated Untreated

C. Anti-ACTIVE® p38 pAbSorbitol-Treated Untreated


6. Composition of Buffers and Solutions

7. References

1. Seger, R. and Krebs, E.G. (1995) The MAPK signaling cascade. FASEB J. 9, 726–35.

2. Cano, E. and Mahadevan, L.C. (1995) Parallel signal processing among mammalianMAPKs. Trends Biochem. Sci. 20, 117–22.

3. Kyriakis, J.M. et al. (1994) The stress-activated protein kinase subfamily of c-Junkinases. Nature 369, 156–60.

4. Gupta, S. et al. (1996) Selective interaction of JNK protein kinase isoforms withtranscription factors. EMBO J. 15, 2760–70.

5. Li, Z. et al. (1996) The primary structure of p38 gamma: A new member of p38 groupof MAP kinases. Biochem. Biophys. Res. Comm. 228, 334–40.

6. Lamarche, N. et al. (1996) Rac and Cdc42 induce actin polymerization and G1 cellcycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.Cell 87, 519–29.

7. Meier, R. et al. (1996) Cellular stresses and cytokines activate multiple mitogen-activated-protein kinase kinase homologues in PC12 and KB cells. Eur. J. Biochem. 236,796–805.

8. Dolmetsch, R.E. et al. (1997) Differential activation of transcription factors induced byCa2+ response amplitude and duration. Nature 386, 855–8.

9. Riesgo-Escovar, J.R. et al. (1996) The Drosophila Jun-N-terminal kinase is required forcell morphogenesis but not for DJun-dependent cell fate specification in the eye.Genes Dev. 10, 2759–68.

10. Sluss, H.K. et al. (1996) A JNK signal transduction pathway that mediatesmorphogenesis and an immune response in Drosophila. Genes Dev. 10, 2745–58.

11. Posas, F. and Saito, H. (1997) Osmotic activation of the HOG MAPK pathway viaSte11p MAPKKK: Scaffold role of Pbs2p MAPKK. Science 276, 1702–5.

12. Payne, D.M. et al. (1991) Identification of the regulatory phosphorylation sites inpp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 10, 885–92.

13. Derijard, B. et al. (1994) JNK1: A protein kinase stimulated by UV light and Ha-Rasthat binds and phosphorylates the c-Jun activation domain. Cell 76, 1025–37.


10% paraformaldehyde5g paraformaldehyde)

50ml PBS

Heat at 75°C with stirring in a fumehood until dissolved. Cool to roomtemperature. Adjust pH to 7.2 with10N NaOH. Add deionized water toa final volume of 50ml.

PVDF buffer

TBS buffer with 0.2% I-Block™(Applera Corporation) and0.1% Tween® 20

TBS buffer20mM Tris-HCl (pH 7.5)

150mM NaCl

TBST bufferTBS buffer with 0.05% Tween® 20


8. Related Products

Product Size Cat. #Anti-ERK 1/2 pAb, Rabbit 40µl V1141Anti-pT183 MAPK pAb, Rabbit 50µl V8081MEK Inhibitor U0126 5mg V1121PD 98059 5mg V1191SB 203580 1mg V1161mNGF, 2.5S 100µg G5141

10µg G5142


© 1998–2009 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 registeredtrademark of Amersham Biosciences. Lab-Tek is a registered trademark of Nalge-Nunc International. Tween is aregistered 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 formore 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 onlinecatalog for the most up-to-date information on Promega products.


mapk, p38 and jnk polyclonal antibodies - promega

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