Hi All,
I'm trying to do a western blot analysis for the detection of Sox-2 in stem cell lines, but i have failed many times over. I kindly need some help and suggestions.
I'm using Sox-2 antibody(Y-17): SC-17320, Santa Cruz. Goat polyclonal anti-human. (1:1000 dilution used)
Secondary was Rabbit anti-goat IgG HRP, Jackson ImmunoResearch. (1:20,000 dilution used).
detection method was by ECL.
Blocking with 5%BSA and 5%skimmed milk gave a heavy background, so tried 5% horse serum and 5% rabbit serum, this resulted in no bands.
I was hoping that 5% rabbit serum would be a good blocking agent, since the secondary Ab was raised in rabbit, then why were there no bands?
Any suggestions are welcome.
Hi, I had to actually order a new lot of the SOX-2 Ab, then things got better.
Pls find the optimised conditions in this open-access paper:
Fatty acid binding protein 7 as a marker of glioma stem cells. Pathology International
Volume 63, Issue 11, pages 546–553, November 2013
I tried the same conditions for WB and ICC, it should work for IHC too, you may need to optimize. Or just make a tissue lysate and do WB.
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