CST - Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb

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REACTIVITY	SENSITIVITY	MW (kDa)	Isotype
H M R Hm Mk Mi Dm Z Pg Sc	Endogenous	42, 44	Rabbit IgG

Western blot analysis of active p42 MAP Kinase, active p38 MAPK and active JNK2 proteins, as well as extracts from PDGF-treated NIH/3T3 cells and EGF-treated A431 cells, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb.

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Immunohistochemical analysis of MDA-MB-453 cells, untreated, EGF-treated or EGF/Erlotinib-treated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human prostate tumor, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb (left) or the same antibody pre-incubated with Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Blocking Peptide #1150 (right).

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Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb.

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Protocol

Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween^® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH₂O, mix.
1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH₂O.
10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH₂O, mix.
10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH₂O, mix.
10X Tris Buffered Saline with Tween^® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH₂O, mix.
Nonfat Dry Milk: (#9999).
Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
Wash Buffer: (#9997) 1X TBST.
Bovine Serum Albumin (BSA): (#9998).
Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
Biotinylated Protein Ladder Detection Pack: (#7727).
Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate.
Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
Microcentrifuge for 5 min.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
Wash three times for 5 min each with 15 ml of TBST.
Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

Wash Buffer: Tris Buffered Saline with Tween^® 20 (TBST-10X) (#9997)
Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H₂0. Make buffer fresh just prior to use.

B. Protocol

After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
Wash membrane six times for 5 min each in TBST.
(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO^® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
Wash membrane again four times for 5 min each in TBST.
The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised October 2016

Protocol Id: 10

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH₂O, mix.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH₂O, mix.

NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
Protein A Magnetic Beads: Use Protein A (#8687) for rabbit IgG immunoprecipitation.
6-Tube Magnetic Separation Rack: (#7017).
10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH₂O, mix.
ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
Sonicate on ice three times for 5 sec each.
Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

Vortex to mix beads.
Add 10-30 µl of 50% Protein A magnetic bead slurry of to 200 µl cell lysate at 1 mg/ml.
Incubate with rotation at 4°C for 30-60 min.
Pellet beads using magnetic separation rack. Transfer the supernatant to a fresh tube.
Proceed to immunoprecipitation below.

Immunoprecipitation

Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
Add protein A magnetic beads (10-30 µl of 50% bead slurry). Incubate with rotation for 10-30 min at 4°C.
Pellet beads using magnetic separation rack. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
Load the sample (15-30 µl) on a 4-20% gel for SDS-PAGE.
Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay

Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
Incubate for 30 min at 30°C.
Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
Transfer supernatant containing phosphorylated substrate to another tube.
Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
Load the sample (15-30 µl) on SDS-PAGE (4-20%).

posted December 2008

revised November 2013

Protocol Id: 410

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

Xylene.
Ethanol, anhydrous denatured, histological grade (100% and 95%).
Deionized water (dH₂O).
Hematoxylin (optional).
Wash Buffer:
1. 1X Tris Buffered Saline with Tween^® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween^® 20 (#9997) to 900 ml dH₂0, mix.
SignalStain^® Antibody Diluent (#8112).
1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
Detection System: SignalStain^® Boost IHC Detection Reagents (HRP, Rabbit #8114).
Substrate: SignalStain^® DAB Substrate Kit (#8059).
Hematoxylin: Hematoxylin (#14166).
Mounting Medium: SignalStain^® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

Deparaffinize/hydrate sections:
1. Incubate sections in three washes of xylene for 5 min each.
2. Incubate sections in two washes of 100% ethanol for 10 min each.
3. Incubate sections in two washes of 95% ethanol for 10 min each.
Wash sections two times in dH₂O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

Wash sections in dH₂O three times for 5 min each.
Incubate sections in 3% hydrogen peroxide for 10 min.
Wash sections in dH₂O two times for 5 min each.
Wash sections in wash buffer for 5 min.
Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain^® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
Equilibrate SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
Remove antibody solution and wash sections with wash buffer three times for 5 min each.
Cover section with 1–3 drops SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
Wash sections three times with wash buffer for 5 min each.
Add 1 drop (30 µl) SignalStain^® DAB Chromogen Concentrate to 1 ml SignalStain^® DAB Diluent and mix well before use.
Apply 100–400 µl SignalStain^® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
Immerse slides in dH₂O.
If desired, counterstain sections with hematoxylin (#14166).
Wash sections in dH₂O two times for 5 min each.
Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 283

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Product Usage Information

Application	Dilutions
Western Blotting	1:1000
Immunoprecipitation	1:100
Immunohistochemistry (Paraffin)	1:800

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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Specificity / Sensitivity

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinase.

Species Reactivity: Human, Mouse, Rat, Hamster, Monkey, Mink, D. melanogaster, Zebrafish, Pig, S. cerevisiae

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Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.

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Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.

1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.

2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.

3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.

4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.

5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.

6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.

7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.

8. Marais, R. et al. (1993) Cell 73, 381-393.

9. Kortenjann, M. et al. (1994) Mol. Cell. Biol. 14, 4815-4824.

10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

Entrez-Gene Id 5595 , 5594

Swiss-Prot Acc. P27361 , P28482

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Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.

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Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb #4376

APPLICATIONS

Gallery: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb #4376

Protocol

Western Blotting Protocol

A. Solutions and Reagents

B. Protein Blotting

A general protocol for sample preparation.

C. Membrane Blocking and Antibody Incubations

I. Membrane Blocking

II. Primary Antibody Incubation

D. Detection of Proteins

Directions for Use:

Western Blot Reprobing Protocol

A. Solutions and Reagents

B. Protocol

Immunoprecipitation for Native Proteins

A. Solutions and Reagents

B. Preparing Cell Lysates

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

Immunoprecipitation

D. Sample Analysis

For Analysis by Western Immunoblotting

For Analysis by Kinase Assay

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

B. Deparaffinization/Rehydration

C. Antigen Unmasking

D. Staining

Product Usage Information

Specificity / Sensitivity

Source / Purification

Background

Data Sheets & Documentation

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