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running problems SDS-PAGE gel (zymography)


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4 replies to this topic

#1 Mieke

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Posted 05 January 2011 - 04:11 AM

Hello everybody,

I am having a problem with doing gel-zymography for quite a while now. The samples don't seem to migrate properly when running my gel. Once they leave the stacking gel, they just stay put and don't migrate further. The ladder does migrate just fine and the loading buffer also moves to the bottom.
Does anyone know a solution for this? (I know people who use the exact same protocol and don't have this problem!)

Many thanks in advance!

Mieke

#2 mdfenko

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Posted 06 January 2011 - 01:34 PM

it sounds like your protein is aggregated.

you used sds-page? if so then you may have overboiled your sample. try boiling for 5 minutes or less or heat at 65-70C for 10-20 minutes.
talent does what it can
genius does what it must
i do what i get paid to do

#3 Mieke

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Posted 11 January 2011 - 07:59 AM

it sounds like your protein is aggregated.

you used sds-page? if so then you may have overboiled your sample. try boiling for 5 minutes or less or heat at 65-70C for 10-20 minutes.


No I did a gel zymography with a 15% gelatin gel, so I don't boil my samples. As a result I see all my bands I want to see but they are packed together on top of my gel.

#4 mdfenko

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Posted 11 January 2011 - 11:17 AM

they may just be large proteins. try a lower percentage, less restrictive gel.
talent does what it can
genius does what it must
i do what i get paid to do

#5 BradH

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Posted 04 June 2012 - 10:25 AM

Hi Mieke,

I am having what sounds like the same problem as you. Did you ever find a solution?

Thanks,
Brad




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