trouble shooting Actin Bands - (Jun/09/2006 )

hi,
i am doing westerns and normalizing my proteins with actin but i have problem with that.i am not getting the same equal bands of actin.some are very dark some are very light and some totally disappears.i am upset becose my boss is not happy with that.pls help me and tell me some reasons and sloutions.thanks

think of you experiment.....maybe actin expression is altered during the course of your experiment...i mean you are trying to use it as internal control but maybe it can't be used as control in your experiment and is affected by the treatment....

try to go for another internal control such as tubulin

how are you quantifying your protein? - try a couple of replicates to make sure that you are actually loading what you think you are loading.....sometimes specs etc are unreliable.....
good luck

QUOTE (saleena @ Jun 9 2006, 10:00 PM)

hi,
i am doing westerns and normalizing my proteins with actin but i have problem with that.i am not getting the same equal bands of actin.some are very dark some are very light and some totally disappears.i am upset with that.pls help me and tell me some reasons and sloutions.thanks

I also use Actin normalizing my protein .... and I have got problem with Actin same as you....
That I'm supposed I think the problem about my lysed procedure and also my sample (normal human fibroblast) passage 12 ... so I prepared early new one ..... and I changed a bit protocol of lysed cells... right now I have a nice signal and my protein signal .... am going to repeat my experimen perhaps something happen ...anyways I hope it could be fine