Western blot problem -disappearing proteins!

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Mon Dec 13 07:15:27 EST 2004


jscorah wrote:

> When we run Western blot gels the proteins and marker go through the stacking 
> gel into 
> the resolving gel and start to resolve nicely. After an hour or so of running 
> the gel the 
> high molecular weight proteins and markers start to disappear from the top 
> downwards. 
> By the time the dye front has reached the bottom of a long gel (16cm gel) the 
> 75kDa 
> marker has started to disappear or become very blurred.

Have you tried to use coloured marker proteins (rainbow-markers or the
like) to watch what happens during the run? 

Where in relation to the front is the 75 kDa protein, i.e. which
percentage gel are you using? Have you considered using gradient gels?
Resolution at the bottom of the gel is quite low unless you use
gradients.

Does the problem also occur with gels bought ready-to-use? 

Do you store the gel o/n after casting it to make sure that all radicals
formed during polymerisation are inactivated?

Is the sample free of salts? Has the acrylamid been stored over ion
exchanger to capture any acrylic acid formed during storage? Measure the
conductivity of sample and gel mixture (before polymerisation) and of
the running buffer. Increased conductivity will lead to band smearing
and gel heating (even boiling!). There are nice pen-shaped conductivity
meters available which are not very expensive and accurate enough for
this purpose, the type I use requires only about 20 ul of sample. 



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