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Western Blotting of Amyloid Beta

Tris-Tricine based PAGE

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#1 Waqar Ahmad

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Posted 20 October 2013 - 07:12 PM

Hi all

I am getting trouble during tris-tricine page. here is image of my gels. I don't know what is the problem. I used all possible ways to eliminate these hurdels but unfortunately nothing work. I need your help to troubleshoot this problem.

 

Regards

Waqar

SBS- University of Queensland

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  • gel.png


#2 mdfenko

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Posted 21 October 2013 - 06:56 AM

a few thoughts...

 

are you sure that you have removed all of the acetone from the pellet?

 

is your sds old? it may be decomposing.

 

have you tried fresh buffers? if so, how well did the old buffers work? the buffers may not have been prepared properly.

 

cold buffers may have crystallized sds present. if you must chill your buffer then use lithium dodecyl sulfate (lds).

 

why are you using a 16% gel? 10% should be sufficient for a 4kDa peptide.

 

do you use stack and spacer gels (your bromphenol blue band is too broad)? they should improve resolution (you can get away without the spacer but not the stack unless you run a gradient gel)


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genius does what it must
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