Overview

  • Product name
    Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody
    See all Erk1 (pT202/pY204) + Erk2 (pT185/pY187) primary antibodies
  • Description
    Rabbit polyclonal to Erk1 (pT202/pY204) + Erk2 (pT185/pY187)
  • Host species
    Rabbit
  • Specificity
    This antibody recognises ERK1 phosphorylated at Thr202 and Tyr204 and ERK2 phosphorylated at Thr185 and Thr187.
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Erk1 (pT202/pY204) + Erk2 (pT185/pY187) conjugated to Keyhole Limpet Haemocyanin (KLH).

  • Positive control
    • NIH3T3 cell lysates stimulated with PDGF.

Applications

Our Abpromise guarantee covers the use of ab16869 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/5000. Predicted molecular weight: 43 kDa.

Images

  • Western Blot analysis of NIH3T3 cell lysates (10µg) stimulated with PDGF

References

This product has been referenced in:
  • Feng M  et al. High glucose increases LPS-induced DC apoptosis through modulation of ERK1/2, AKT and Bax/Bcl-2. BMC Gastroenterol 14:98 (2014). WB, IHC . Read more (PubMed: 24885625) »
  • Velten M  et al. Systemic maternal inflammation and neonatal hyperoxia induces remodeling and left ventricular dysfunction in mice. PLoS One 6:e24544 (2011). WB ; Human . Read more (PubMed: 21935422) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

Thank you for your patience in this matter.

I am very pleased to inform you that the product (positive control lysate for ab16869 - NIH3T3 cell lysate stimulated with PDGF) is now available and we are now doing the final internal necessary steps. Though, it is not for general sale. Once the datasheet is created, we can ship the product to Sapphire.

Let me know if you have any further questions.

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Answer

Thank you for your kind reminder.

I will be sending an e-mail as soon as have further information about the lysate from the Lab.

Thank you for your patience in advance.

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Answer

I can now inform you that we do not have the lysate in stock currently. This is expected to be available at the earliest next month.

I am sorry for the inconvenience. If you have any further questions or concerns please do not hesitate to contact us.

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Answer

Thank you for your enquiry regarding ab16869.

The cells need to be stimulated and in order to be able to detect phosphorylated ERK1 + ERK2 PDGF is recommended as inducer.

Your customer may wish to take a look at this specific publication:

Velten M et al. Systemic maternal inflammation and neonatal hyperoxia induces remodeling and left ventricular dysfunction in mice. PLoS One 6:e24544 (2011). WB; Human.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21935422&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21935422&dopt=Abstract

If you need any further assistance in the future, please do not hesitate to contact me.

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Application
Western blot
Sample
Mouse Cell lysate - whole cell (BV2 cell line)
Loading amount
400000 cells
Specification
BV2 cell line
Treatment
LPS 50 ng/mL or LPS 50 ng/mL + UO 10 uM
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 08 2008

Application
Western blot
Sample
Rat Tissue lysate - whole (spinal cord homogenate)
Loading amount
20 µg
Specification
spinal cord homogenate
Treatment
sham or nerve transected
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 08 2008

Application
Western blot
Sample
Rat Cell lysate - whole cell (HAPI cell line)
Loading amount
400000 cells
Specification
HAPI cell line
Treatment
LPS 50 ng/mL or LPS 50 ng/mL + UO 10 uM
Gel Running Conditions
Reduced Denaturing (10 % SDA-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jun 11 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (Primary Microglia)
Loading amount
400000 cells
Specification
Primary Microglia
Treatment
LPS 50ng/mL or LPS 50 ng/mL + UO 10 uM
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Ms. Nancy Nutile-McMenemy

Verified customer

Submitted Jun 09 2008

Question

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Rat bladder smooth muscle cell total protein extracts PRIMARY ANTIBODY Primary antibody was ab16869 for phospho erk 1/2 produced in rabbit. Antibody was used at a ratio of 1:5000 in the first trial and 1:1000 in the second trial. Antibody was diluted in a solution consisting of: 2 grams of blotto dry milk dissolved in PBS/Tween solution at a total volume of 100 mL. Primary antibodies were incubated for 1 hour followed by 3-15 minutes rinses in the PBS/Tween solution. DETECTION METHOD Chemiluminescence using the BIORAD developer kit and the Alpha Innotech FluorChem SP imager. POSITIVE AND NEGATIVE CONTROLS USED Positive control consisted of bladder smooth muscle cells exposed to human recombinant IGF for 45 minutes. Negative control consisted of bladder smooth muscle cells without any stimulation. ANTIBODY STORAGE CONDITIONS Antibodies were stored in aliquots in the -20 C freezer. SAMPLE PREPARATION Cells were removed from dishes by scraping and collected by centrifugation at 300 x g. Pellet was resuspended in ice cold PBS and again centrifuged at 300 x g. PBS was removed and cells were lysed in a cell extraction buffer consisting of: 100 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, and a protease inhibitor cocktail. Mixture was incubated on ice for 30 minutes with occasional vortexing (every 10 minutes). Lysates were clarified by centrifugation at 14,--- rpm at 4 C for 10 minutes. AMOUNT OF PROTEIN LOADED In the first trial, 40 micrograms of protein were loaded into each lane of the Western blot. In the second trial, 58 micrograms of protein were loaded into each lane. ELECTROPHORESIS/GEL CONDITIONS SDS-page gel was used with the acrylamide percentage at 12%. Unsure whether the gel was reducing or non-reducing. TRANSFER AND BLOCKING CONDITIONS Transfer was carried out at 100 V for 1 hour using the BIORAD transfer buffer. Protein transfer was confirmed using Ponceau staining. Membranes were blocked overnight at 4 C using a blocking solution consisting of: 10 mL 10x PBS, 100 uL 10% Tween 20, 10 grams of blotto dry milk, and brought to 100 mL using DH2O. SECONDARY ANTIBODY Secondary antibody was anti-rabbit produced in goat by Santa Cruz Biotechnology. The antibody was HRP-conjugated. It was diluted in the same diluent as above (5 uL of the secondary antibody in 40 mL of the PBS/Tween solution). Secondardy antibodies were incubated for 1 hour followed by 3-15 minute rinses in the PBS/Tween solution. (I know this secondary antibody works because I used it with my GAPDH primary antibody and am able to visualize the bands.) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? The only things I have changed between trial is the amount of protein loaded per well and the dilution of the primary antibodies. ADDITIONAL NOTES I know that the protein transfer is successful because I stained the membrane with Ponceau following transfer. Also, I was able to successfully label the membrane with other HRP-conjugated primary antibodies such as: alpha smooth muscle actin, GAPDH, and SM-22. Therefore, I know the issue is not with the secondary antibodies. Thank you for your help.

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Answer

Thank you for filling out our technical help questionaire so thoroughly. You protocol looks correct, however I advise for this or any other antibody against a phosphorylated protein that you avoid using milk in your diluents and blocking buffer. Milk contains casein, a phosphorylated protein which antibodies against other phospho-proteins tend to bind to. Substituting BSA should improve you results. You may also get a stronger signal if you incubate the primary antibody with the membrane for longer than one hour, for instance overnight at 4C. If these suggestions do not help, please do not hesitate to contact us.

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Answer

Thank you for contacting us with your query. There is only a small difference between these two antibodies if you are going to make a choice between the two. They will both recognise phosphorylated ERK1 and ERK2. ERK1 and ERK2 are phosphorylated within the activation loop on both a Threonine and a Tyrosine residue (within a Thr-Glu-Tyr motif) by MEKs (MAPK/ERK kinases), thereby greatly elevating their activity . Ab4819 recognises phosphorylation on the T185 and T202 residues Ab16869 recognises phosphorylation on T202/T183 and Y204/185 residues. These phosphorylated residues are common to ERK 1 and 2. Both the antibodies are raised against synthetic peptide. I hope this helps! Please get back to me if there is anything else I can help you with. With thanks.

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