Western blot troubles - Bands diffuse

[elisangelasantos]

I have eight groups to compare with Western blot and my band of interest appears, but the bands are diffuse and form one line. I already did western putting smaller amount of protein and another with a lower voltage (90V), but still cannot separate my bands. What should I do?

[mdfenko]

instead of using a 8-10 slot comb, use a 15 or more slot comb and use alternating wells (fill the intervening wells with blank samples).

you may be able to reduce diffusion if you load less volume and/or run the gel faster.

[toxlab]

Since you have eight groups, that is tough to run in fewer lanes and fewer wells...You did not mention how much protein you have loaded in the original or lower amount gel...for some targets, you can run very little protein, like 5 ug. If you are running much higher than this, I would try reducing it greatly to avoid the lane spillover effect.

[relaxin]

I do not think protein overloading is the problem. I use 50 ug protein per lane, I got a thick band on Western, but the bands are not touching each other. Did you prepare the gel one day ahead? Perhaps the "ears" (the small piece of gel separating the wells) of your gel have dried and allow the samples leak into the neighboring lanes.

[mdfenko]

another thing,

do you let the gel sit after loading, before starting the run or do you load slowly? either may allow the sample to diffuse towards the adjoining lanes.

your problem is more likely due to the composition of the samples' buffer (not the sample loading buffer). salts, surfactants and other additives can affect the way in which your samples run.