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SDS PAGE smearing of bands


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#1 biotef

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Posted 23 June 2011 - 08:49 PM

Hello All,

We are facing a peculiar problem.

I have attached my SDS PAGE gel for reference. We get a pattern as if protein has spilled into adjacent well and the blank well (1st and last) also shows band. we have been performing the same method for same protein for about 7 yrs with no difficulty but from past 2-3 months are encountering this problem. we have tried preparing fresh reagents and do the test but same results. what can be the problem as while loading the protein does not spill, still this results is confusing.

Can anyone throw some light or elaborate on this. Any help will be appreciated.

Thanks in anticipation

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#2 protolder

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Posted 26 June 2011 - 10:16 PM

Hello All,

We are facing a peculiar problem.

I have attached my SDS PAGE gel for reference. We get a pattern as if protein has spilled into adjacent well and the blank well (1st and last) also shows band. we have been performing the same method for same protein for about 7 yrs with no difficulty but from past 2-3 months are encountering this problem. we have tried preparing fresh reagents and do the test but same results. what can be the problem as while loading the protein does not spill, still this results is confusing.

Can anyone throw some light or elaborate on this. Any help will be appreciated.

Thanks in anticipation

Hola, Probably if you load less sample and avoid to fill the well untill the top you will avoid the problem, if you do it Ill try to add a bit more of acrylamide in the stacking buffer. Buena suerte

#3 mdfenko

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Posted 15 July 2011 - 07:53 AM

you may have another problem, besides overloading.

you are seeing a buffer front with your overloaded protein. this can be caused by a number of factors including salt in the sample and the condition of buffer components.

if the electrode buffer was adjusted for pH then you may have salt present.

the sds may be old and, at least partially, decomposed (the powder as well as the liquid).

try preparing fresh electrode buffer with fresh components.

also, fill any unused well with diluted sample buffer.

Edited by mdfenko, 15 July 2011 - 07:54 AM.

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