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15% SDS-PAGE issues


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#1 nnady

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Posted 23 April 2013 - 02:00 PM

I'm trying to run 15% SDS-PAGE to check for histones and am having issues with that. I'm making my own gels and have made 10% gels which look and run normal. For whatever reason the bottom portion of a 15% gel is empty, literally, the sample (which is also fuzzy) only is becoming visible half through the gel. I'm very puzzled since my 10% gels look fine. ANy suggestions?

#2 sikander

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Posted 23 April 2013 - 07:32 PM

Hiii

i wanna suggest yo uthat you should check the pH of the buffers. It really matter.


good luck

#3 mdfenko

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Posted 24 April 2013 - 04:09 AM

a 15% gel is very restrictive and takes longer to run than lower percentage gels.

rather than running for a specific length of time, follow the tracking dye and stop the gel when it reaches the bottom. if you are already doing that and seeing the dead space then it may be due to your sds. try a new lot of sds (not a fresh preparation of the same lot).

why did you switch to 15% when you obtained good results with 10%?

if it is to improve resolution then you may want to try a 12% gel or a 10-15% gradient.

or a 10% tris-tricine gel.

Edited by mdfenko, 24 April 2013 - 04:10 AM.

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

#4 nnady

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Posted 24 April 2013 - 07:40 AM

thanks for your replies, I'll try to make fresh Tris pH8.8 and 6.8 as well as find another batch of SDS. I have to run higher percentage 15% or 18% to resolve histones (~15kda). I tried running the gel at a very low voltage for longer time and it doesn't make a difference and yes, I always follow the dye front and not just the time. I've been running 15% gels for years in my old lab (biochemistry lab where I did my PhD), but now just started my post-doc in a cell bio/stem cell lab and they are very very rarely running 15% gels.

#5 mdfenko

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Posted 25 April 2013 - 04:38 AM

15kDa is right in the sweet spot of a 10% tris-tricine gel (schagger and von jagow, they also came up with blue-native page). you might want to consider it. i've found it to be very useful for the resolution of smaller proteins and peptides (including fragments from proteolysis).

Edited by mdfenko, 25 April 2013 - 04:40 AM.

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




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