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Areas of membrane bleached - (Jul/24/2011 )

Dear All,

I have been running western blots in the last year, in the same lab, using the same reagents from the same companies, the same machines, buffers, protocol, etc. I have never experienced any problems until recently when the following problems started.

Let me explain what I am doing. I am detecting, for example AKT and b-actin antibodies (2 antibodies ordered from the same company, raised in the same species, requiring the same blocking and primary antibody solutions). I have always used the following procedure: 1h blocking in dry milk, overnight incubation with the primary antibody at room temperature (in 5% BSA), secondary antibody (dry milk for 1h RT), detection with West Dura/Femto substrate (with 1xTBST washes in between each treatment). On the first day, I detect, for example, AKT and I have no problems - the blot looks perfect!. Then, i block again the membrane and incubate overnight with, for example, the b-actin protein. This procedure has always worked perfectly until now. What happens now is that on the next day part of the membrane is bleached or some blank areas appear, which makes the quantification impossible (see picture1). Or another problem I had was that the lanes in the center of the membrane were looking good, while the lanes on the sides of the membrane were lighter (see picture2). Ponceauís dye shows equal transfer. Furthermore, i have always exposed the membrane for 120sec first and then for 300sec to get clearer results. What happens now is that after the first exposure, the signal is either completely lost, or the membrane looks even more bleached until the signal is completely lost in the end.

I have tried preparing new buffers, incubating with new antibodies, making sure that the volume of the primary antibody solution is enough and equally covering the membrane during the overnight incubations, but nothing solved the problem. I have given you example with p-AKT and b-actin here, but the same thing happens when using other antibodies as well - one day everything works perfect, on the next day the membrane is completely messed up. A colleague of mine started having the same problems recently - the only thing we share with him is the chemiluminescent substrate.

Do you have any suggestions?
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It really looks like you have a problem with one of the solutions not covering the blot - it could be secondary or even the detection reagent. Did the membranes dry out at all between blots?

If you do more washes between primary and secondary (3-4 10 minute washes in TBST is usual) you will cut down your background.