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Why Squeezing of lanes occur at bottom in PAGE


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#1 biochemscientist

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Posted 22 September 2013 - 08:29 PM

I am encountring another problem in 4-13% gradient native gel. The gradient gel is prepared perfectly indicated by the marker bands which are very appropriate in proper lane. The problem lies in the sample lane which from the top are perfect but from the bottom they are some what squeezed what might be the reason behid this as i have some bands at the lower end.

 



#2 Curtis

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Posted 22 September 2013 - 10:40 PM

low gel percentage could be one of the reasons. also try to stop the gel when the dye reaches the bottom of the gel next time.



#3 mdfenko

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Posted 23 September 2013 - 03:46 AM

are there any additives in the sample buffer (eg non-ionic surfactants, peg, etc), salt?

 

more information (and a picture, if possible) is necessary to make an accurate diagnosis.


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#4 biochemscientist

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Posted 23 October 2013 - 02:07 AM

mdfenko here is the picture which i am getting the current ones are even more squeezed from the bottom. it is native PAGE

Attached Thumbnails

  • photo0618.jpg


#5 mdfenko

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Posted 23 October 2013 - 04:13 AM

it's not just the pinching at the bottom. it's also the blob above the pinch.

 

these effects are caused by additives in the buffer in which you have your sample.

 

what is the buffer (just to make sure, sorry if this is insulting but has happened in the past, you're not making your samples up in laemmli sample buffer?)?

 

i've seen these effects with detergents, peg, salt...


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#6 biochemscientist

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Posted 23 October 2013 - 12:53 PM

The sample solution contains NaCl, Imidazole/HCl, 6-Aminohexanoic acid, EDTA, glycerol, and coommassie blue. since it is native PAGE not SDS PAGE.

 

Reference: Blue Native PAGE (Wittig I, Braun HP, Schagger H) Nature Protocols



#7 mdfenko

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Posted 25 October 2013 - 04:26 AM

what is the source of your sample? if it is cell or tissue culture then the "blob" may be due to albumin from residual serum (i thought someone else had posted this but it appears to have been deleted).

 

have you tried native page (not blue native, blue native modifies the protein and may even denature it)?

 

the sample solution you posted has a few components which may effect the appearance of the gel (depending on concentration and pH).


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