Anti-Integrin alpha 4 antibody [44H6] (ab220)

Which portion of the integrin alpha4 that the antibody will detect? Is it the extracellular domain, or cytplasmic domain?

ANSWER:

Thank you for your enquiry.

This product recognises the extracellular portion of integrin alpha 4. However, since epitope mapping was never done, there is no information on the exact epitope sequence.

I'd like to know if this antibody recognizes the N-term region of alpha 4 integrin or which part of the protein. Thanks in advance. Best regards

ANSWER:

Thank you for your enquiry regarding ab220. We have not mapped the epitope region recognized by this antibody therefore we do not know which region is recognized by the antibody.

Sorry I couldn't help you more on this occasion,

follow up to previous enquiry: The two different positive controls were histag versions of human integrin alpha 4, both were loaded at approximately 50ng. It is possible there isn't enough protein in my samples but that doesn't explain why I'm not detecting my positive controls. Can you suggest a good starting dilution for this antibody also?

ANSWER:

Thank you for your email. We have had feedback from a customer who used this antibody at a 1:200 dilution for Western blotting. I'm really not sure why this antibody is not working for you, and can offer (if you have purchased the antibody in the past 90 days) a replacement vial to try or a refund or credit note. Please let me know how you would like to proceed.

follow up to previous enquiry:

To answer your questions reqarding inquirey 735273 1. My transfer was good because my molecular weight markers transfer fine and I ran a number of gels at the same time and they all worked fine (different antibodys) 2. I found the lot number and it is 146463 3. I don't know the amount if any protein was loaded, I am determing mammalian cell transfection of the recombinant integrin alpha 4 from cell culture supernatant and am trying to verify expression.

ANSWER:

Thank you for your email. I noticed in your previous email that you mentioned for a positive control, "tried 2 different previously verified positive controls." Can you please elaborate on that? For your other samples, the soluble integrin alpha 4 secreted into cell supernatant, I wonder if there's enough present there to be detected by this antibody.

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

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Soluble integrin alpha 4 secreted into cell supernatant, lacking c-terminus which is very small

PRIMARY ANTIBODY AB220 mouse anti-human 60ul/15mls BSA==4ug/ml from a 1mg/ml stock Incubated overnight at 4C Wash in TBST 15min. 3 times

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Yes, human integrin alpha 4/His

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION 1X SDS running buffer heated 4 min.

AMOUNT OF PROTEIN LOADED trying to determine

ELECTROPHORESIS/GEL CONDITIONS Reducing gel 5-20%

TRANSFER AND BLOCKING CONDITIONS Transfer 1 hour at 200V Block with 3% dry milk and no results Also tried BSA and no bands Have numerous good positive controls and none are showing up

SECONDARY ANTIBODY goat anti-mouse HRP 1:2000 dilution in BSA 1 hr. Room Temp. Wash 3X for 15 min.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Blocking reagents: tried 3% milk alone and BSA alone. Positive control: Tried 2 different previously verified positive controls

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. Did you determine how much protein was loaded onto the gel? Also, do you know if the protein transferred properly to the membrane?

What was the batch number (located on the vial) or the Abcam order number or purchase order number that was used when you purchased ab220?

Thank you, and I look forward to hearing from you.