I am lysing the QT6 cells transfected after 48 hrs and doing my SDS-PAGE and western blotting. My protein has the HA antigen tag and i use 1:1000 dilution of HA monoclonal Ab.
To troubleshoot this problem v 2 people tried with the same blot, the same procedure and the same buffers. But i am getting again with high background. But the other person result was with clear band (only my protein of interst) without any bg.
It is difficult to identify where things r going wrong with my handling.
1) 1 hr blocking with 5% skim milk prepared in 1X TBS-T(tween 20 0.05%)
----after this, mild wash with 1X PBS-T to remove traces of skim milk.
2) 1 hr primary ab(1:1000) prepared in 1X TBS-T
3) wash 3 times with 10 min duration each with 1X TBS-T
4) 1 hr Sec ab (1:1000) prepared in 1X TBS-T
5) wash 3 times with 10 min duration each, with 1X TBS-T
6) ECL -Amersham Reagent and developing.
Expecting a reply
PVDF membranes? Make sure they are correctly activated with methanol...not doing so will give black background.
is primary polyclonal? try larger dilutions if yes...1:5000 - 1:10000.
1:1000 secondary is HRP conjugate? This sounds too high. 1:5000 to 1:20000 depending on quality of the primary.
Wash more at all washing steps. Heed the advice already offered about "pre-washing" extra Ab before your timed washes. I have never over-washed a blot in my life and I'll wash sometimes 4-5 changes 20 min. each only because I am running too many other experiments at the same time.