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Anti-Blooms Syndrome Protein Blm antibody (ab476)

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If your product does not perform as described on this datasheet, we will refund or replace your product...

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3 questions for ab476

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Question 1

Monday 15-April-2002

Does the anti-blm antibody detect _mouse_ blm on western blots? If so, what dilution and blocking conditions work best?

ANSWER:

  

As stated on the datasheet, "Cross reactivity with blm from other species has not been tested but is highly likely against mammalian homologues of human blm."

Question 2

Wednesday 21-March-2001

The antibody works well in detecting BLM protein in human fibroblasts. I am currently attempting an IP. Since the antibody is sera with no known conentration what dilution/volume of antibody should I use in an IP?

Thank you,

ANSWER:

  

For an IP, use 0.5 - 5 µl antiserum.

Question 3

Monday 12-February-2001

HI.. we have a customer who purchased the anti-BLM antibody and tested it on HeLa cell lysates. He sees the Bloom band but several other non-specific bands. He is wondering the conditions you used to get the single band in your blot.

Thanks

Karen

ANSWER:

  

The blot shown on the datasheet is using HeLa Nuclear extracts, not whole cell extracts. However, there are several things to bear in mind:

1- It may be possible to dilute the antibody a bit more: We have used it at 1:2500 with good results.

2- Blm is very sensitive to degradation... so extracts must be done carefully. A useful control could be cells that are lysed directly in a buffer containing 6 M Urea, 20 mM Tris ph 7.5, sonicate for 10 seconds, and centrifugue for 5 min at 13000rpms in an micro-centrifugue (and take the supernatant). If you make extracts for IPs, the protein can be easily degraded, with this control you avoid that.

3- Sometimes it helps to strip the blot before blocking (and, of course before the incubation with the primary antibody).

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