Anti-CD44 antibody (ab24504)

I am currently using your ab24504, CD44 antibody in my research. My question is what variant or isoform of CD44 does this antibody recognize?

ANSWER:

Thank you for your enquiry.

At this point we do not have any data from testing the different isoform specificity of this antibody.

All of our products are guaranteed to work in species and application as stated on the datasheet. Should you not get expected results, then please inform us within the guarantee period of 6 month (Abpromise). We will help troubleshoot the experiment and if the product is indeed faulty, we will be more than happy to offer a free replacement or refund.

I hope our Abpromise guarantee will give you the assurance you need to use this product in your research.

What should be used as a positive control in WB?

ANSWER:

Thank you for your enquiry. HeLa cell extract can be used as a positive control with this antibody.

Please contact us again if you have any additional questions.

Thank you for your prompt response. I attached one of my results to you. As you can see, there are no significant band at 82 KD (predicted). There are only heavy chain shown up in IP samples because of long time exposure. This is a SDS Phage, we do not think there are problems for our transfer. When i did IP, i added 30 ul (6 ug) of antibody for 300 ul MEF lysate ip which is in the range of the suggestions of your company. I believe somebody already test this antibody before your company selling it. So could you show me your company's result of Western and IP and conditions as well. Although communicating with you is good, i still do not want waste too much time on this matter. If this still can not convince you, please test this antibody on MEF cells or any cell lines which express CD44 by your company and give me a convincing result and showing this antibody is good.

ANSWER:

Thank you for getting back to me with an image of your blot.

Once again I am sorry to hear that you have been having difficulties with this antibody. Your image is very convincing and the fact that you are not observing a band of 82KDa from a murine lysate is very telling. This is despite the prolonged exposure of your blot. Indeed the large number of extraneous bands that you have obtained in the embryo lysate is of some concern.

Quality is important to Abcam. Given that this antibody has not behaved as detailed on our datasheet against your mouse lysates, I would like to offer you a credit note provided that the antibody was purchased within the past 90 days. For me to do this please can you e-mail me details of the date of purchase and original purchase order number.

I look forward to hearing from you.

BATCH NUMBER 149473 ORDER NUMBER 118536 DESCRIPTION OF THE PROBLEM Can not detect any bands SAMPLE Mouse embryonic fibroblast PRIMARY ANTIBODY ab 24504 ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION Ripa MATRIX USED Protein A/G DETERGENT SDS solution AMOUNT OF PROTEIN LOADED the lysis coming from one 10 cm plate ELECTROPHORESIS/GEL CONDITIONS Reducing 10 % SECONDARY ANTIBODY Protein A/G -hrp HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES We use the same method done so many times IP. If no special reason to explain this failure, we thought this ab does not work at all. We can not just depend on your seggestions to test this Ab again and again, so please give me your good protocol or please test this Ab by yourself.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody by IP. I noticed that you have also made an enquiry about this antibody by western blotting. Quality is important to Abcam. I would appreciate it if you could provide me with the details that I mentioned in my previous e-mail regarding further information of the conditions that you have been using by western blotting. Should this antibody not work by western, I consider it possible that it will not work by immunoprecipitation.

With regards your IP conditions, please can you tell me how much antibody that you have been using. This antibody is supplied quite dilute (0.2mgml-1) with a recommended dilution of 20ugml-1 by IP; therefore a 1:10 dilution is required. I would also be interested in the wash conditions that you have been using and whether you could quantitate the mass of protein that you have been using. We recommend 200ug - 500ug RIPA lysate.

I appreciate your patience in this matter and look forward to your response.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Can not detect 82 KD perdicted band, but i can see some not right size weak bands. SAMPLE Mouse embryonic fibrolast, cell extract. PRIMARY ANTIBODY ab 24504 DETECTION METHOD ECL plus ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION Ripa AMOUNT OF PROTEIN LOADED 100% cofluency10 cm plate cells in 300 ul Ripa and load 10 ul ELECTROPHORESIS/GEL CONDITIONS reducing 10 % TRANSFER AND BLOCKING CONDITIONS over 2 hrs HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody.

I have read your questionnaire and I have a few comments. It would greatly help me if you could provide me with a quantitation value of the mass of protein that you are loading. I would like to recommend that you quantitate the protein and load 20-40ug for analysis by western blotting.

It would also assist me if you could provide me with details of how the samples were prepared for electrophoresis. Did you use denaturing (SDS) conditions in addition to reduced conditions? Were the samples prepared using Laemmli buffer? Did you verify the integrity and successful transfer of the protein by using a Ponceau stain of the membrane?

Also could you please provide me with details of the secondary antibodies you have used. Finally I would appreciate it if you could attach an image of the blot with the molecular weight markers marked.