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442704 Anti-MAP Kinase ERK1/ERK2 Rabbit pAb

This Anti-MAP Kinase ERK1/ERK2 Rabbit pAb is validated for use in Flow Cytometry, Immunoblotting, Paraffin Sections for the detection of MAP Kinase ERK1/ERK2.

Anti-MAP Kinase ERK1/ERK2 Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
442704
  
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Overview

Replacement Information

Key Spec Table

Host
Rb
Description
OverviewRecognizes the ~42-44 kDa ERK1 and ERK2 proteins.

This product has been discontinued.
Catalogue Number442704
Brand Family Calbiochem®
Application Data
Detection of rat MAP kinase ERK1/ERK2 by immunoblotting. Samples: Whole cell extracts from serum-induced PC12 cells. Primary antibody: Anti-MAP Kinase ERK1/ERK2 Rabbit pAb (Cat. No. 442704) (1:1000). Detection: chemiluminescence.
References
ReferencesHill, C.S. and Treisman, R. 1995. Cell 80, 199.
Hunter, T. 1995. Cell 80, 225.
Marshall, C.J. 1995. Cell 80, 179.
Cowley, S., et al. 1994. Cell 77, 841.
Product Information
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 100 µg/ml BSA, 50% glycerol, pH 7.5.
PreservativeNone
Applications
Key Applications Flow Cytometry
Immunoblotting (Western Blotting)
Paraffin Sections
Application NotesFlow Cytometry (1:25)
Immunoblotting (1:1000)
Paraffin Sections (1:100)
Application CommentsVariables 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% bromophenol 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 PC12 cells per well in a 6-well plate is as follows:

1. Culture cells in medium containing 0.5% FBS for 2 days. We recommend plating cells directly in 0.5% FBS media to reduce basal levels of MAP kinase phosphorylation.
2. Aspirate media. Add fresh 0.5% FBS media. Culture for 2 h.
If cells are grown at high density, changing media before treating cells with regulator reduces basal MAP kinase phosphorylation due to factors secreted by the cells.
3. Aspirate media. Treat cells by adding fresh 0.5% FBS media containing regulator for desired time.
4. Aspirate media from cultures; wash cells with PBS; aspirate.
5. Lyse cells by adding 100 µl SDS Sample Buffer per well and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
7. Heat sample to 95-100°C for 5 min. Cool on ice.
8. Microcentrifuge for 5 min.
9. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
10. Electrotransfer to nitrocellulose membrane.
As controls, we recommend using 10 µl of phosphorylated (Cat. No. 454855) and nonphosphorylated
(Cat. No. 454852) MAP kinase control proteins.

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 of 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 of 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 of 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 synthetic peptide corresponding to amino acids from rat p42 MAP kinase, conjugated to KLH
ImmunogenHuman
HostRabbit
IsotypeIgG
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
Catalogue Number GTIN
442704 0

Documentation

Anti-MAP Kinase ERK1/ERK2 Rabbit pAb MSDS

Title

Safety Data Sheet (SDS) 

Anti-MAP Kinase ERK1/ERK2 Rabbit pAb Certificates of Analysis

TitleLot Number
442704

References

Reference overview
Hill, C.S. and Treisman, R. 1995. Cell 80, 199.
Hunter, T. 1995. Cell 80, 225.
Marshall, C.J. 1995. Cell 80, 179.
Cowley, S., et al. 1994. Cell 77, 841.
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.

Revision07-August-2007 RFH
ApplicationFlow Cytometry (1:25)
Immunoblotting (1:1000)
Paraffin Sections (1:100)
Application Data
Detection of rat MAP kinase ERK1/ERK2 by immunoblotting. Samples: Whole cell extracts from serum-induced PC12 cells. Primary antibody: Anti-MAP Kinase ERK1/ERK2 Rabbit pAb (Cat. No. 442704) (1:1000). Detection: chemiluminescence.
DescriptionRabbit polyclonal antibody purified by protein A and immunoaffinity chromatography. Recognizes the ~42-44 kDa ERK1 and ERK2 proteins.
Backgroundp44 and p42 MAP kinases (ERK1 and ERK2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation. MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (amino acids 202 and 204 of human MAP kinase (ERK1) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK)).
HostRabbit
Immunogen speciesHuman
Immunogena synthetic peptide corresponding to amino acids from rat p42 MAP kinase, conjugated to KLH
IsotypeIgG
Specieshamster, human, mouse, rat, zebrafish
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 100 µg/ml BSA, 50% glycerol, pH 7.5.
PreservativeNone
CommentsVariables 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% bromophenol 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 PC12 cells per well in a 6-well plate is as follows:

1. Culture cells in medium containing 0.5% FBS for 2 days. We recommend plating cells directly in 0.5% FBS media to reduce basal levels of MAP kinase phosphorylation.
2. Aspirate media. Add fresh 0.5% FBS media. Culture for 2 h.
If cells are grown at high density, changing media before treating cells with regulator reduces basal MAP kinase phosphorylation due to factors secreted by the cells.
3. Aspirate media. Treat cells by adding fresh 0.5% FBS media containing regulator for desired time.
4. Aspirate media from cultures; wash cells with PBS; aspirate.
5. Lyse cells by adding 100 µl SDS Sample Buffer per well and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
7. Heat sample to 95-100°C for 5 min. Cool on ice.
8. Microcentrifuge for 5 min.
9. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
10. Electrotransfer to nitrocellulose membrane.
As controls, we recommend using 10 µl of phosphorylated (Cat. No. 454855) and nonphosphorylated
(Cat. No. 454852) MAP kinase control proteins.

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 of 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 of 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 of 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
ReferencesHill, C.S. and Treisman, R. 1995. Cell 80, 199.
Hunter, T. 1995. Cell 80, 225.
Marshall, C.J. 1995. Cell 80, 179.
Cowley, S., et al. 1994. Cell 77, 841.