just a few technique questions.
1.) Do you sterile filter all reagents (TBST, blocking buffer, etc.) before use
2.) How long do you wash for each wash cycle? I've been washing 5 minutes each 3x for each wash step - is this excessive?
3.) When diluting antibodies, how do you ensure equal mixing? Do you pipette up and down? Do you vortex?
4.) How long do you block for? How long do you apply your antigen/sample/analyte? I've been doing two hours at RT. I'm using cell lysate and was wondering if I need to put a protease inhibitor in the solution if incubating for two hours.
Sorry for all of the questions, I know some have been covered elsewhere but I'm finding a lot of conflicting information between different sources!