Sox-2 antibody detection using Western - (Oct/27/2011 )
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.
did you try milk as blocking agent?
yes, but got a heavy background
did you try changing your prmary antibody or secondary antibody dilution?
no i did not, maybe i need to increase the secondary antibody concerntration..
For your BSA/milk blocking system, if there is a high background then may be you can try optimize the primary/secondary dilutions. I just had this problem a few weeks ago, so what I did was run a gel of the exact same sample, and then cut off each lane and then try out different combinations of primary and secondary dilutions, and see if it gets better.
why did you use horse serum?
try a rabbit serum/bsa mix (use use host of secondary/bsa mix to block our blots).
honestly, i tried horse serum bcoz that was the next best thing i got in the lab before i could get some rabbit serum.
i also learned that since goat and cow are phylogenetically close, BSA or milk for blocking interacted with the rabbit anti-goat secondary. however, i also learned from yesterday's blot that horse serum though not interacting with the secondary Ab, created too many non-specific band. today as suggested by zienpiggie im trying out different dilutions of secondary and will stick to rabbit serum.
thanx a lot for the comments.
Greetings from Santa Cruz.
I see you are having trouble using your Sox-2 antibody for WB. Did you already contact Technical Support here at Santa Cruz? We warranty all our products for 1 year from the date of purchase for the species and applications listed. We would be glad to help you try to get this Western Blot to work for you. If you want to contact us and share your protocol and results with us, we would be glad to offer suggestions to your protocol for this antibody or if we see necessary, we can replace it with a different lot (if we have one available) or another Sox-2 antibody.
Please feel free to give us a call or send us an email and we will be glad to help you. You can reach us in several ways:
Toll Free: 1.800.457.3801 ext. 2
Good luck in your research!
Santa Cruz Technical Support Team
Santa Cruz Biotechnology, www.scbt.com
Phone: +1-800-457-3801 ext. 2
Thanks you Santa Cruz Technical Support Team for spotting me here,
Yes I have contacted the Technical Support, and have submitted a trouble shooting sheet with results, and was told that it will take ten days for processing since im in Japan.
however I shall also mail you the results in detail.
this is awesome help!