Millipore Sigma Vibrant Logo
Attention: We have moved. EMD Millipore products are no longer available for purchase on emdmillipore.com.Learn More

559304 Anti-SAPK/JNK Rabbit pAb

This Anti-SAPK/JNK Rabbit pAb is validated for use in Immunoblotting, Immunocytochemistry for the detection of SAPK/JNK.

Anti-SAPK/JNK Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
View Products on Sigmaaldrich.com
559304
Purchase on Sigma-Aldrich

Overview

Replacement Information

Key Specifications Table

Species ReactivityHostAntibody Type
H, M, RRbPolyclonal Antibody

Products

Catalog NumberPackaging Qty/Pack
559304-200UL Plastic ampoule 200 ul
Description
OverviewRecognizes the ~54 kDa SAPK/JNK protein in uv-treated HEK293 cells.
Catalogue Number559304
Brand Family Calbiochem®
Application Data
Detection of human SAPK/JNK by immunoblotting. Sample: Whole cell lysate from HEK 293 cells and SK-N-MC cells treated with UV (40 J/m2) for the indicated times. Primary antibody: Anti-SAPK/JNK Rabbit pAb (Cat. No. 559304) (1:1000). Detection: chemiluminescence.
References
ReferencesGupta, S., et al. 1996. EMBO J. 15(11), 2760.
Coso, O.A., et al. 1995. Cell 81, 1137.
Derijard, B., et al. 1994. Cell 76, 1025.
Kyriakis, J.M., et al. 1994. Nature 369, 156.
Hibi, M., et al. 1993. Genes Dev. 7, 2135.
Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.
Product Information
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
Positive controlUV treated HEK293 cells
PreservativeNone
Quality LevelMQ100
Applications
Key Applications Immunoblotting (Western Blotting)
Immunocytochemistry
Application NotesImmunoblotting (1:1000)
Immunocytochemistry (1:200)
Application CommentsRecognizes SAPK/JNK regardless of the phosphorylation state. Variables associated with assay conditions will dictate the proper working dilution.

Recommended Protocol for Immunoblotting

Solutions and Reagents
•Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5.
•SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue.
•10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
•Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
•Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
•Wash Buffer (TBST): 1X TBS, 0.1% Tween®-20 detergent

Blotting Membrane
Nitrocellulose or PVDF membranes may be used.

Protein Blotting
A general protocol for sample preparation using 2x106 293 cells per well in a 6-well plate is as follows:

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with PBS; aspirate.
3. Lyse cells by adding 100 µl of SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
4. Sonicate for 2 s to shear DNA and reduce sample viscosity.
5. Heat sample to 95-100°C for 5 min. Cool on ice.
6. Microcentrifuge for 5 min.
7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
8. Electrotransfer to nitrocellulose membrane.

As controls, we recommend using 20 µl lysate from UV treated HEK293 cell.

Membrane Blocking, Gel and Antibody Incubations
1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
3. Wash 3 times for 5 min each with 15 ml TBST.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
5. Wash 3 times for 5 min each with 15 ml TBST.
6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
7. Wash membrane as in step 5.

Detection of Proteins
Chemiluminescence.
Biological Information
Immunogena full-length, recombinant, human p54 SAPK/JNK2 fusion protein
ImmunogenHuman
HostRabbit
IsotypeIgG
Species Reactivity
  • Human
  • Mouse
  • Rat
Antibody TypePolyclonal Antibody
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage -20°C
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Catalog Number GTIN
559304-200UL 04055977192421

Documentation

Anti-SAPK/JNK Rabbit pAb SDS

Title

Safety Data Sheet (SDS) 

Anti-SAPK/JNK Rabbit pAb Certificates of Analysis

TitleLot Number
559304

References

Reference overview
Gupta, S., et al. 1996. EMBO J. 15(11), 2760.
Coso, O.A., et al. 1995. Cell 81, 1137.
Derijard, B., et al. 1994. Cell 76, 1025.
Kyriakis, J.M., et al. 1994. Nature 369, 156.
Hibi, M., et al. 1993. Genes Dev. 7, 2135.
Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision15-August-2007 RFH
ApplicationImmunoblotting (1:1000)
Immunocytochemistry (1:200)
Application Data
Detection of human SAPK/JNK by immunoblotting. Sample: Whole cell lysate from HEK 293 cells and SK-N-MC cells treated with UV (40 J/m2) for the indicated times. Primary antibody: Anti-SAPK/JNK Rabbit pAb (Cat. No. 559304) (1:1000). Detection: chemiluminescence.
DescriptionProtein A and immunoaffinity purified rabbit polyclonal antibody. Recognizes the ~54 kDa SAPK/JNK protein.
BackgroundThe Stress-activated protein kinases (SAPK), also referred to as Jun N-terminal kinases (JNK), function in a protein kinase cascade transducing cellular stress signals. SAPK/JNKs are activated by a highly diverse group of extracellular signals, including UV light, inflammatory cytokines and a wide variety of cellular stresses. Activation occurs via phosphorylation at Thr183 and Tyr185 by the dual specificity enzyme SEK/MKK4 and as yet unidentified kinases. Since dual phosphorylation of SAPK/JNK at Thr183/Tyr185 is essential for kinase activity, phosphorylation at this site is an excellent marker of SAPK/JNK activity. Activated SAPK in turn phosphorylates and activates several transcription factors, including c-jun at Ser63/73 and ATF-2 at Ser69/71.
HostRabbit
Immunogen speciesHuman
Immunogena full-length, recombinant, human p54 SAPK/JNK2 fusion protein
IsotypeIgG
Specieshuman, mouse, rat
Positive controlUV treated HEK293 cells
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
PreservativeNone
CommentsRecognizes SAPK/JNK regardless of the phosphorylation state. Variables associated with assay conditions will dictate the proper working dilution.

Recommended Protocol for Immunoblotting

Solutions and Reagents
•Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5.
•SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue.
•10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
•Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
•Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
•Wash Buffer (TBST): 1X TBS, 0.1% Tween®-20 detergent

Blotting Membrane
Nitrocellulose or PVDF membranes may be used.

Protein Blotting
A general protocol for sample preparation using 2x106 293 cells per well in a 6-well plate is as follows:

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with PBS; aspirate.
3. Lyse cells by adding 100 µl of SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
4. Sonicate for 2 s to shear DNA and reduce sample viscosity.
5. Heat sample to 95-100°C for 5 min. Cool on ice.
6. Microcentrifuge for 5 min.
7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
8. Electrotransfer to nitrocellulose membrane.

As controls, we recommend using 20 µl lysate from UV treated HEK293 cell.

Membrane Blocking, Gel and Antibody Incubations
1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
3. Wash 3 times for 5 min each with 15 ml TBST.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
5. Wash 3 times for 5 min each with 15 ml TBST.
6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
7. Wash membrane as in step 5.

Detection of Proteins
Chemiluminescence.
Storage Avoid freeze/thaw
-20°C
Do Not Freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
Toxicity Standard Handling
ReferencesGupta, S., et al. 1996. EMBO J. 15(11), 2760.
Coso, O.A., et al. 1995. Cell 81, 1137.
Derijard, B., et al. 1994. Cell 76, 1025.
Kyriakis, J.M., et al. 1994. Nature 369, 156.
Hibi, M., et al. 1993. Genes Dev. 7, 2135.
Kyriakis, J.M. and Avruch, J. 1990. J. Biol. Chem. 265, 17355.