Anti-CD95 antibody (ab2437)

Hope you had a great 4th of July weekend. Thanks so much for looking into my enquiry.

I'm pretty sure my marker is fine as other proteins appear at the right molecular weight, including your abcam mouse GAPDH loading control.

The samples are tissue culture whole cell lysates made using a very standard method. The Jurkat control whole cell lysate was purchased commercially from Santa Cruz, so I don't think our cell prep method is off.

I will look into your other suggestions however.

ANSWER:

It is my pleasure, please let me know if you require additional assistance regarding this antibody.

Attached please find a western blot using the replacement vial of ab2437. As you can see, I have beautiful bands in the Jurkat control and my samples. The size seems to be closer to 60 kDa than 44 kDa as described in your literature. What do you think? Do I have a Fas band?

ANSWER:

Thank you for your email and patience. I contacted the originator of this antibody in order to get some additional feedback regarding your results. With ab2437 and Jurkat whole cell lysate, the originator sees a band at approximately 50 kDa (as you can see from the image on the datasheet) and there are reports in the literature reporting that FAS/CD95 ranges in size from 45-50 kDa. So this is consistent with those reports.

It is a possibility that your molecular weight markers may be off. You could try a different FAS/CD95 antibody for comparison. We also do sell the blocking peptide (catalog number ab2438) for ab2437 - you could use this in order to determine whether the band you are seeing is specific.

The originator also did say "The only other reference to observing FAS at 60 kDa is soluble FAS/CD95 binding serum FASL and when they blotted the samples they reported seeing it at 60 kDa. I do not know how the samples were produced/collected or if it is even possible for FASL to be present to bind with FAS/CD95."

I hope this helps. If you need additional assistance, please let me know. Have a great weekend!

Jen, Please send a replacement vial. I'll try again. Thank you!!!

ANSWER:

Thank you for your email. The replacement vial of ab2437 is being sent free of charge to your attention on order# 88169 and you should receive this tomorrow. Please let me know if you have any additional questions and how the replacement works out for you.

Dear Jen,

Last week I used the CD95 Ab @ 1:100 dilution; incubated @ 4 degrees overnight. I also used ~ 100 ug of protein per lane. I had imaged the blot using a BioRad Chemi Doc. I had to really overexpose to see any bands. I do see a faint band @ 60 kDa. Is this Fas?

At the moment I am reprobing with one of your GAPDH Ab's to check my loading. To answer the storage question, I used the Ab within 24 hrs of arrival. It has been stored @ 4 degrees which I believe is okay for short term storage.

This email is just to keep you up to date on teh situation. I may call you later.

ANSWER:

Thank you for your email. With ab2437 you should see a band at approximately 50 kDa - as shown in the Western blot image on the datasheet using it at 1:200 with Jurkat whole cell lysate. You stored the antibody correctly -it should be stored at 4C and not frozen. If you would like, I can send you a replacement vial.

Customer is using this antibody in Western blotting with Jurkat and A-431 lysates at a dilution of 1:200 but is not seeing any bands. Secondary antibody is working fine.

Abcam lot# 68540 Order# 83831

ANSWER:

Thank you for your phone call. I contacted the originator of this antibody and there have not been any problems reported with this lot. Were there any improvements when you increased the dilution to 1:100? Did you incubate overnight at 4C? How did you store the antibody upon receiving it?

Thanks again, and I look forward to hearing from you.