Anti-Notch1 antibody [EP1238Y] (ab52627)

I have tested this antibody in WB and observe no bands. I would like to test another Notch1 antibody.

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 ab27526.

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.

Hi, Thanks for your response. Does this protocol specifically isolate plasma membrane or is it all cellular membranes (i.e. organelle membranes as well)?  

ANSWER:

Thank you for your reply. This protocol would isolate the plasma membrane and organelle membranes as well. 

http://www.abcam.com/index.html?pageconfig=resource&rid=11473

If you're looking to only isolate the plasma membrane, you'd have to use our Plasma Membrane Extraction Kit, ab65400.

http://www.abcam.com/Plasma-Membrane-Protein-Extraction-Kit-ab65400.html

I hope this information helps.  Please contact us with any other questions.

We've been using the Notch1 antibody (ab52627) on total cell lysate from HEK293T cells and had great results by Western Blot. The product details cite HEK293 Plasma membrane lysate as a positive control and as it so happens, we are very interested in the expression in the Plasma Membrane Lysate. Can you recommend a good protocol for isolating just the plasma membrane from these cells for use with this antibody?

ANSWER:

Thank you for your inquiry. We have a protocol for cell fractionation that can be found here:

http://www.abcam.com/index.html?pageconfig=resource&rid=11473 I hope this information helps. 

Please contact us with any other questions.  

WB with human kidney cancer, pancreatic cancer and breast cancer lysates sees strong band around 50-60 kDa, but nothing at 120 or 250 kDa loaded up to 100 ug protein Ab: 1/1500 overnight 4 degree block: ready-to-use reagent from Pierce/Thermofisher for 1h secondary: works fine

ANSWER:

Thank you for contacting us. I am sorry to hear that this antibody is not working as expected.

As discussed over the phone this morning, please let me know which of the 2 antibodies (ab27526 or ab8925) you would prefer to receive as free of charge replacement.

Just to let you know, other options would be to receive a credit or refund or a replacment with an antibody to a different protein of your choice.

I look forward to hear back from you and resolve this issue to your satisfaction.

WB with human cancer cell lines

sees consistent band at 110-120 kDa

predicted MW is 270 for full length unprocessed precursor

sent database link and explanation

ANSWER:

Thank you for contacting us. The database entry for the human NOtch1 protein is as follows: http://www.uniprot.org/uniprot/P46531

There you will find more information about the protein structure, sequences and additional references.

In regards as to why you see band at 110-120 kDa: The predicted MW is 270 kDa for the full length unprocessed precursor. If the protein has splice variants or can be cleaved, than shorter versions of the protein can be detected - if they contain the immunogen sequence (cytoplasmic portion). According to the database entry, the protein can get cleaved into Notch 1 extracellular truncation and Notch 1 intracellular domain. The antibody would be able to recognize then the intracellular domain. However, under which condition the cleavage happens, I am not certain. I would suggest to consult the literature for this detailed information.  

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.