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:
  • Liu CY  et al. Varlitinib Downregulates HER/ERK Signaling and Induces Apoptosis in Triple Negative Breast Cancer Cells. Cancers (Basel) 11:N/A (2019). Read more (PubMed: 30658422) »
  • Wang J  et al. LINC00707 promotes hepatocellular carcinoma progression through activating ERK/JNK/AKT pathway signaling pathway. J Cell Physiol 234:6908-6916 (2019). Read more (PubMed: 30317590) »
See all 58 Publications for this product

Customer reviews and Q&As

1-8 of 8 Q&A

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.

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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.

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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.

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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.

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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.

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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.

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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,

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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

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Answer

DISCOUNT CODE: xxxxxx Expiration date: 2012-04-01 I am very pleased to hear you would like to test ab50011 in locust. This testing scheme applies to any untested species or application not listed on the datasheet for the antibody which you are interested in. If you then purchase the antibody, test this new a application or species, then share your results in the form of an Abreview you can get your next antibody free. The testing discount does need to be generated before purchase however. More detailed information can be found here: www.abcam.com/collaborationdiscount The code above will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for locust with ab50011 and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview (should only take 5-10 min), please visit this site:  www.abcam.com/Abreviews Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests whatever the results. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. We are also currently running a promotion which may be of interest to you with the same concept but this time it is not for an untested application or species but for an image. You can apply for a testing discount where we do not currently have an image displayed on the datasheet. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

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Answer

For the detection of total ERK1+2 we recommend ab17942, a Rabbit polyclonal directed against amino acids 317-339 of Human ERK1+ERK2. This region is not subject to phosphorylation. ab17942 is covered by our Abpromise guarantee to work in ICC/IF, IHC-Fr, IHC-P, IP, WB and Human, Mouse, Rat, Cow, Dog. For the detection of the phosphorylated ERK1+2 we recommend ab50011, a Mouse monoclonal specific for the active, dually-phosphorylated form of MAP kinase (ERK-1 and ERK-2, 44 kDa and 42 kDa, respectively). It does not recognise the non-phosphorylated or the monophosphorylated forms of the MAP kinase molecule or the diphosphorylated forms of JUN kinase and p38 MAP kinase. 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). ab50011 is guaranteed to work in ELISA, ICC/IF, IHC-P, IP, WB and Human, Mouse, Rat, Cow, Drosophila, yeast. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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