I am treating RMC cells (Rat Kidney cells) with various concentrations (100ul, 250ul, and 500ul) of apocynin. After 24 hours I use to prepare western sample and I can hardly see the variations among these different concentrations. As some earlier paper shows band intensity should be increase from lower to higher concentration of apocynin.
I am using primary antibody 1:250ul (abcam) with 5% skim milk. Here abcam protocol suggest a concentration of 4ug/ml.
I use to protein sample concentration up to 90ug/ml.
Can anybody suggest what could be the problem with my settings?
Anti-inflammatory activities of apocynin may be by the inhibition of the assembly of NADPH-oxidase that is responsible for reactive oxygen species (ROS) production.
So, i suggest that try to increase the day of treatment to 48hrs and then check between 24hrs and 48hrs sample. Also, treat with primary antibody at 3% skim milk and 5% BSA.
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