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poor seal between stacking a seperating gels


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#1 kaffs

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Posted 01 August 2011 - 08:12 PM

Hi,

I've been running Western blots for about 2 years and I've just started having a problem which I can't find any trouble shooting information on.
I am casting my own gels, making 1mm thick minigels with an 8% seperating gel and a 5% stacking gel. I make up my APS fresh each time. Recently I have started to have issues with the join between the two gels. When I load my samples they run through the stacking gel, but when they reach the interface between the two gels, there is very rapid lateral spreading along the join, as if there is a gap. they then proceed to run normally through the seperating gel. I have looked carefully at the gels before running and couldnt see anything unusual about the interface. I have transfered one of these gels, and immunoblotted it. The ponceau S showed that there was protein staining between the lanes. When I immunoblotted it, my actin bands ~45kDa) had joined, however the bands for my target protein (which runs at ~68kDa)were clean and distinct.
Has anyone else had a similar problem with a poor join at the interface? I am being very carefull when I remove the comb. The only thing I can think of is that perhaps my stacking well wasn't deep enough, so its association with the glass isn't strong enough to stop them from pulling apart

Any help would be hugely appreciated! I am frantically trying to finish off some Westerns before I handin my thesis in 4 weeks!

Thanks in advance

#2 mdfenko

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Posted 02 August 2011 - 08:38 AM

you may be pulling the comb out too harshly.
talent does what it can
genius does what it must
i do what i get paid to do

#3 bob1

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Posted 02 August 2011 - 05:26 PM

What do you overlay with while the separating gel is setting?

If it is butanol, you need to wash the gel top off with water before adding the stacker.

#4 kaffs

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Posted 02 August 2011 - 05:53 PM

thanks for your replies. I am overlaying with ddH2O, and drying the glass with filter paper before pouring the stacking gel.

#5 powertoeki

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Posted 13 August 2011 - 12:55 AM

Did you happen to check the pH of stacking and the resolving buffers? How frequently do you make them? How much more water can you possibility remove from between those two layers?

Check pH of the sample, or the loading dye.

Also, when you remove the water using filter paper, put the casting gel like 90 degree (tilt the casting gel system) to collect most of the water with filter paper.

I think I used to have similar problem when pH of stacking and resolving buffers were not checked by phD student... ( he used to be lab tech and he never measured pH of those solutions.. .)




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