Hi everyone.
I would like to know if someone have already used anti-GAPDH (G5259) to western blot. I tried once diluted 1:10000 in 5% low fat dried milk/TBS-T for 1 hour in RT. The same way I use actin, but it did not work.
Is there any tip to help?
Thank you.
Fernando
Assuming that you had appropriate controls (positive and negative) for your experiment, it is likely that you need to optimize conditions. Most antibodies are diluted 1:1,000 times. Further, you did not indicate whether you "macrophages" were murine or human and whether you use total cell extracts or sub-cellular fraction(s). The link below could help you in optimizing conditions to detect GAPDH in immunoblotting.
Link: http://www.jbc.org/content/282/5/3252.full
Hi Fernando, We routinely use this particular antibody in the lab for probing westerns. We as a rule of thumb load 10ug of protein on a gel and then probe with the Ab for 2h at RT in Skim milk at 1:2500 dilution. Also make sure your secondary is working properly. Hope this helps.
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