western blot, I have a signal without primary antibody - (Nov/16/2008 )
Hi I have been using ECL mix to detect the optimum primary Antibody concentrations for a western blot and found out that even without the primary antibody I have a signal for protein blots.
Can anyone shed some light? please!
Did you use secondary antibody on the blot?
yes, I did two blots with the primary antibody and without the primary antibody. both blots gave exactly the same intensity signal.
Seems like your secondary ab is binding to some proteins..
Are you blocking the membrane properly?
yes the blocking reagent is part of ECL kit. I block it at RT for 1 hour.
this must be the answer.
what is your sample anyway? cell lysate?