Hi all, I've just got in an Abcam HIF-1a Ab with the intention of running both Western Blots (frozen pig brain tissue) and Immuno (PFA fixed, paraffin embedded tissue). Just wanting to hear from anyone who has worked with the #AB463 antibody before, & what WB and IHC conditions/blocking solutions and Ab dilutions did you use?
Also happy to hear from anyone whose done same techniques with AQ4 and MMP9.
Hi, I have worked with HIF1-alpha for Westernblot and the dilutions are as follow:
Blocking soln: 10%
Primary Ab: 1:500
Secondary Ab: 1:1000
Good luck!
Thanks Shabnam, and your preps were in a lysis buffer with standard protease inhibitors?
Cheers, S
Your welcome. I am glad I could help Stephanie. My preps were as you said. You know, if HIF1-alpha is expressing low in your samples, it would be better you try either 2 hr or overnight probing with primary antibody.
Looks like you are working on HIF-1 alpha mAb’s Clone H1alpha67. From Novus, we offer customized protocols for IHC-P, ICC/IF and WB analysis using HIF1 mAb’s H1alpha67 clone: http://www.novusbio.com/HIF-1-alpha-Antibody-H1alpha67_NB100-123.html
For troubleshooting tips and more than 20 FAQs on HIFs & Hypoxia, I would suggest reading: http://www.novusbio.com/support/hypoxia-and-hif-faqs
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