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problem with sds-page - (May/08/2014 )

Hi

1. Due to an unknown reason, low molecular weight  bands in my gel have low resolution.

I use fresh buffers.

Because the pH of Tris solutions is temperature-dependent, I adjust the pH of the solutions at room temperature and use them at the same temperature. 
The attached file is my gel picture.
What do you think about the reason? 
Attached Image
 2. Why sometimes proteins dont create a thin zone in the stacking gel with a length of 1 centimeter?

-marzieh-

What percentage gel are you running and what size are you after?

-bob1-

Thanks for your attention. 

I use 15% resolving gel and my protein MW is about 8 KD.

-marzieh-

with laemmli sds-page, low molecular weight proteins are difficult to maintain sharp banding. just using high percentage acrylamide normally doesn't help. a gradient may improve the sharpness but we find that the best method for sharp separation of low molecular weight proteins and peptides is that of shagger and von jagow (tris-tricine sds-page). a 10% acrylamide gel should give you sharp banding of your 8 kDa protein.

 

however, sometimes lower molecular weight band sharpness is affected by aged sds. you can try preparing buffers with a fresh lot of sds and see if it improves sharpness.

 

but, i think you'll get better results in general if you switch to tris-tricine-sds.

 

ps. you should find the formulation by searching the forum.

-mdfenko-

Thank you very much.

In the cases where lower bands are not sharp, sometimes i encounter lack of concentration of the protein sample in the 1 cm stacking gel. What do you think about the reason?

-marzieh-

In the cases where lower bands are not sharp, sometimes i encounter lack of concentration of the protein sample in the 1 cm stacking gel. What do you think about the reason?

only if your sample volume is too large to stack properly.

-mdfenko-

Thanks for your helps  smile.png

-marzieh-

let us know what you do and how it works

-mdfenko-

hi.

the problem was partially solved by using fresh sds

-marzieh-