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Can Current affect to the samples in the edge/periphery of the gel?


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#1 Utsav

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Posted 15 July 2011 - 01:50 PM

I have run almost twelve vertical gels in Bio-Rad Mini Tank till this date. Everytime, I have used 10 well combs in order to run eight different samples of my experiment. I always put marker in the first lane from left side, and then in the following four lanes I load my control samples which were treated with different AICAR (a pharmacological agent) time course from 0 min. to 60 min. (order of samples in gel is 0 min., 10 min. , 30 min., and 60 min. treated samples from left to right) and after that in another four lanes I load my experimental samples which were also treated with different AICAR time course from 0 min. to 60 min. (same order of samples in gel as of control) and then in the last lane of right side I load 1X sample buffer as a blank.

I was examining my films since from few days. During that I have come across one surprising thing. I have observed that almost in six experiments out of twelve, with the second lane from the left menas the lane in which I have loaded 0 min. AICAR treated control sample and with the eighth well from the left means the lane in which I have loaded my 60 min. AICAR treated experimental sample show less amount of beta actin protein and histone H3 too (which are like housekeeping proteins, express equally in any condition) compare to other lanes. So, I have thought two possible reasons for that:

1.) 60 min. AICAR treatmnt may affect on the whole cell protein level (I have other supporting data for this thought so I am not worried ablut this) OR
2.) Is it possible that current may affect on the edge / periphery of the gel especially in the last two lanes from both sides?

I am less thinking for defects in transfer procedure because all the experiments have shown me pretty good cncluding results with different antibodies.

So, the whole idea to give the compendium of my experiments is that I wanna know that current which I apply to the gel, I run my samples at 200 V and 60 mAs, can affect to the whole protein content especially only in the last two lanes from both sides.

#2 bob1

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Posted 16 July 2011 - 09:53 PM

No it will not affect the lanes separately - the current should be evenly distributed over the gel if the electrode wires in the buffer chambers are connected properly.

You can test the effect you are seeing very simply by switching the position of those two samples for different ones.

It is more likely that you are seeing some antibody effect by not evenly immersing the membrane in the antibody diluent (you should have enough to coat the membrane evenly, and should shake the membranes in this solution at about 60 rpm to ensure that you are getting even coating).




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