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SDS-Page trouble-shooting

sds-page coomassie

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#1 Bacillus_Guy

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Posted 29 July 2013 - 01:03 AM

Hey everybody,

 

attached you will find protein gel I just made and I have some problems to fix:

 

You see that the protein lanes get first thinner and second more blurry the smaller the proteins are.I have no idea how to fix that?

 

I've read some threads saying salt concentrations might be an issue. Right now, I'm dialysing my samples to see if it helps, but i do you think it will prevent that the proteins lines become thinner?

 

I used a discontinuos SDS-PAGE gel with 12.5%, the samples are a whole cell extract from a bacterial culture (B. subtilis)

 

Thank you in advance for any help I can get!

 

Best,
Bacillus_Guy

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#2 mdfenko

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Posted 29 July 2013 - 04:32 AM

salt is as good a guess as any.

 

are the gels homemade or commercial? if commercial, how old? if homemade, how old is the sds (not just the solution)?

 

the "blurring" may be due to decomposed sds although you will often see more diffusion of the bands as they travel through the gel.

 

if not caused by decomposed sds (you can test by using a new lot of sds) you can reduce blurring by running a gradient gel.

 

but, first see if dialysis helps.


talent does what it can
genius does what it must
i do what i get paid to do

#3 Bacillus_Guy

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Posted 30 July 2013 - 01:00 AM

Ok, first thanks for the replying so fast!

 

Desalting helped a bit, yet I must say, bands are still blurry (I'll upload the gel as soon as destaining is finished)

So, the gels are homemade, the 1x running buffer is usually not older than a week (the 10x buffer stock might be 2 month old) and the 10% SDS I use to prepare the gels is a couple of months old, do you think that is the problem?

I'll try to fix it and let you know...



#4 mdfenko

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Posted 30 July 2013 - 03:48 AM

even the powdered sds will decompose over time. you should prepare a fresh solution with a new lot of sds (you may be able to borrow powder or solution from a fresh lot from another lab) to determine if that is the cause of your problem.


talent does what it can
genius does what it must
i do what i get paid to do





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