Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [MAPK-YT] (ab50011)

Overview

  • Product name
    Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [MAPK-YT]
    See all Erk1 (pT202/pY204) + Erk2 (pT185/pY187) primary antibodies
  • Description
    Mouse monoclonal [MAPK-YT] to Erk1 (pT202/pY204) + Erk2 (pT185/pY187)
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, ELISA, WB, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Saccharomyces cerevisiae, Drosophila melanogaster
    Predicted to work with: Xenopus laevis, Zebrafish
  • Immunogen

    Synthetic peptide corresponding to Erk1 (pT202/pY204) + Erk2 (pT185/pY187) conjugated to Keyhole Limpet Haemocyanin (KLH). Synthetic peptide corresponds to the phosphorylated form of human ERK1 and ERK2 activation loop.
    Sequence:

    HTGFLpTEpYVAT

  • Epitope
    The epitope recognized by ab50011 is located in the region of phosphorylated threonine and tyrosine residues within the regulatory site of active MAP kinase (Thr185 and Tyr187 in ERK-2).
  • Positive control
    • Rat brain extract, Drosophila wing imaginal disc.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 25th October 2016. The following lot is from ascites and is still in stock as of 25th October 2016- GR185138. Lot numbers higher than GR185138 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

Properties

Applications

Our Abpromise guarantee covers the use of ab50011 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
ICC/IF 1/200.
ELISA Use at an assay dependent concentration.
WB 1/10000. Predicted molecular weight: 44 kDa.
IP Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

Images

  • Lane 1 : Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [MAPK-YT] (ab50011) at 1/10000 dilution
    Lane 2 : no primary antibody

    All lanes : Extract of Rat1 cells activated with 100µM
    Vanadate+200µM H2O2

    Secondary
    All lanes : Goat Anti-Mouse IgG, AlkPhos
    Conjugate

    Predicted band size: 44 kDa
    Observed band size: 42,44 kDa
    why is the actual band size different from the predicted?

  • In situ immunostaining of the activated form of MAP kinase with ab50011 (green) in Drosophila wing imaginal disc (500 micrometers long).
  • ICC/IF of NRK cells labeling the activated form of MAP kinase with ab50011 (1/1000 dilution). The antibody was developed using Goat Anti-Mouse IgG, Cy3™ conjugate. Cells were counterstained with DAPI (blue).

References

This product has been referenced in:
  • Oh SC  et al. Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype. Nat Commun 9:1777 (2018). Read more (PubMed: 29725014) »
  • Su L  et al. Leptin attenuates the growth of rabbit mesenchymal stem cells via the extracellular signal-regulated kinase signaling pathway. Exp Ther Med 15:4185-4190 (2018). Read more (PubMed: 29731817) »
See all 49 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Zebrafish Tissue lysate - whole (Total protein from zebrafish testicle)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
10 µg
Treatment
Animals exposed to bisphenol A during 21 days
Specification
Total protein from zebrafish testicle
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 15 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Xenopus laevis Tissue sections (Tadpole limb)
Permeabilization
Yes - 1% Triton X-100
Specification
Tadpole limb
Blocking step
Roche blocking reagent as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Fixative
Formaldehyde

Gufa Lin

Verified customer

Submitted Sep 12 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (skin)
Permeabilization
No
Specification
skin
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Aceton methanol

Abcam user community

Verified customer

Submitted Feb 06 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Dako REALPeroxidase-Blocking Solution S2023 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
Antigen retrieval step
Heat mediated
Sample
Zebrafish Tissue sections (Liver, dysplatic cells)
Specification
Liver, dysplatic cells
Permeabilization
No
Fixative
Paraformaldehyde

Dr. Inhye Jung

Verified customer

Submitted Jul 17 2014

Answer

Thank you for your reply and further question.

This monoclonal antibody in the ascites fluid that was produced by the hybridoma within the mouse peritoneal cavity. As far as I can tell there should only be the specific antibody present in terms of immunoglobulins, however as this is a (relatively clean) bodily fluid, other mouse proteins will be present.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact me.

Read More

Answer

Thank you for your enquiry.

I can confirm that the total protein concentration in ab50011 should be between 15 - 55 mg/ml when measured by Biuret and that the IgG1 concentration should be between 3 - 6 mg/ml.

Regrettably we do not have any other information regarding affinity and antibody immunogen interactions. Unfortunately, it is unusual to have this type of information for research grade antibodies.

I am sorry we do not have all the information you require on this occasion, however I hope it is still helpful. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement of a different lot number with the order number 1140603.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

Thank you for your reply.

I am very sorry to confirm that we sell a lot of this antibody and regrettably we now have no vials of GR121-7 left.

As previously discussed, I can confirm that our lots GR121-7 and GR121-8 are the same original batch from the producer, the vials have just come to us in different deliveries. I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably it seems you received a bad vial on this occasion.

However, I do fully understand your concerns, and would be pleased to provide you with a free vial from a completely different lot to try. Or I can provide a credit note.

I am sorry your prefered batch ran out of stock so quickly. Thank you for your understanding. I look forward to hearing from you with details of how you would like to proceed in this case.

Read More

Question

Order Details
Antibody code: ab50011
Lot number:GR121-8
Purchase order number
or preferably Abcam order number: Purchase order number is ####
General Information
Antibody storage conditions (temperature/reconstitution etc)
Stored at 40C
Description of the problem (high background, low signal, non-specific
satining etc.)
This batch gave no nuclear staining as expected, when we worked up the
Ab using GR121-7. All staining was cytoplasmic with a small percentage
of membranous.
Sample (Species/Tissue/Cell Type/Cell Line etc.) Human colorectal
tumour tissue
Fixation of sample
(Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Formalin fixed, and paraffin embedded.
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Vector Antigen unmasking solution, 2 minutes in a pressure cooker
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.) 15
minutes in hydrogen peroxide, and 10 minute is 1x casein block
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
Ab50011 GR121-8, 1 in 50 dilution, diluted in Zymed Antibody diluents
containing 1:50 dilution of casein. Incubated 16 hours at 4 degrees C.
Washed off with TBS.
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
DAKO envision plus mouse secondary HRP-polymer, for 30 minutes. Again
washed off with TBS.
Detection method
DAB, followed by counterstaining with Hematoxylin and lithium carbonate.
Positive and negative controls used (please specify)
Human kidney sections used.
Optimization attempts (problem solving)
How many times have you tried the IHC?
We optimised lot number GR121-7, over several runs, changing dilution
factor, retrieval method, secondary Ab type etc, but when GR121-8 was
run using the identical conditions used to optimise GR121-7, we saw NO
nuclear staining as expected, and previously demonstrated. We have
repeated the GR121-8 run and saw exactly the same staining pattern as
seen previously, where NO nuclear staining was detected.
Have you run a "No Primary" control?
Yes
Do you obtain the same results every time?
Yes
What steps have you altered? NO steps were altered between runs using
GR121-7 and GR121-8, so we expected to have obtained identical
staining patterns.
Additional Notes
We would appreciate if you are also able to provide and image which
would help us to assess the results
We have attached 2 images, one of GR121-7 and GR121-8. Identical
sections were stained with both antibodies, and you can quite clearly
see the difference between the 2 images.

Read More
Answer

Thank you for taking the time to complete our questionnaire and provide the images.

The details you have kindly provided will provide us with vital information for our monitoring of product quality.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggestthat regrettably youmayhavereceived a bad vial.

I can recommend you may like to consider trying the following options which may help provide more consistent results between batches:

1. I can recommend to try serum to block rather than casein. Changing blocking agents can sometimes help to improve results.

2. I can suggest to wash in PBS containing 0.2% Tween. The Tweenwill help to keep the cells permeabilised, and the antibody solubilised.

3. I can recommend toaliquot and store at -20oC or -80oC as directed on the datasheet. This will help to ensure the antibody remains stable and that we can continue to provide our guarantee.

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More

Question

As discussed over the phone earlier today, we have encountered a problem with the Abcam pERK Ab.

Our initial working up of this was carried out using Ms mAb to ERK1 +ERK2, [MAPK-YT] (phospho T185), 50ul (5.2mg/ml), AB50011, Lot: GR121-7.

The results of this were good, reasonably clean, nuclear staining on our colorectal cancer TMA sections and control kidney sections.

We determined the optimal dilution to be 1:50.

The TMA image can be viewed at http://129.11.191.7/Research_4/Clinical_Trials/Susan_Richman/Gaya/190712/view.apml?

The file is 183507.svs and can be opened with Webscope or Imagescope if you download the free software.

I should be able to send you an image of the slides stained with lot number GR121-8 tomorrow or Monday as a comparison.

AS we had insufficient Ab left, I re-ordered it, and was sent a vial identical to the above, with the exception of the lot number being GR121-8, instead of -7.

The resultant staining with this new lot showed absolutely no nuclear staining whatsoever. There was minimal membranous staining at best. The protocols used were identical in every way, except the Ab.

This is a real concern as there is obviously lot to lot variations and we have unfortunately wasted valuable clinical trial material. Have such problems been encountered elsewhere?

We are limited by time on this project, so hope to resolve this ASAP. Would you be able to look into sourcing more of the lot GR121-7 for us, which we could re-test on colorectal control material before sacrificing valuable clinical material again.

Look forward to hearing from you in due course,

Read More
Answer

Thank you for taking the time to contact us. You enquiry has been forwarded to the scientific support team. I am sorry to hear you have had difficulty obtaining satisfactory results from this vial of antibody.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

In answer to your question regarding the lot numbers, we do have lot GR121-7 still in stock. However, reviewing the details I can confirm that our lots GR121-7 and GR121-8 are the same original batch from the producer, the vials have just come to us in different deliveries. I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch.

I would also like to reassure you that this antibody is tested and covered by our 6 monthguarantee for IHC-P (paraffin embedded) and human samples. Before deciding how to proceed, I would like to investigate this particular case further for you, and also obtain some further information for our quality records.

In order to do this, I have enclosed a questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. I would appreciate if you are able to provide some images which will help us to assess the results. It would be helpful if these could be attached to the reply email in PDF or JPEG format.

In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which would help us to assess the results

Read More

1-10 of 21 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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