Hi, can any one suggest me, i am performing western blot to detect tnf alpha in brain tissue. i am using 12%gel and wet transfer method, tried with NC and PVDF. When i stain the gel i could do the separation of protein and b actin also working but with tnf alpha there is no bands in membrane. so what could be the reason.
could it be because TNF it is secreted? Have you considered doing an ELISA of the extracellar fluid?
TNF is unstable but I suggest using a positive control for the western.
1. definitely load positive control and Milk as blocking agent for 18 hour(s)· Concentration: 5% · Temperature: 4°C. some people also use Pierce TBS Super Block as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 22°C
2. use Reduced Denaturing from 9% to 12 % SDS-PAGE.
3. the band might appear as 17 kDA for some tissues
4. the Anti-TNF alpha antibody (ab6671) from Abcam seems the best.
@ Franco Grimolizzi - Thnku, Ya i have totally 8 groups which include positive control as wel
@ Olga Sukocheva - Thnku, 1. You mean to increase my blocking time to 18 hrs, so far i kept for 1.30hrs
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