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Tenfold PBS in extraction


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#1 Mirandish

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Posted 05 October 2011 - 06:49 AM

By mistake I extracted my proteins with 10x PBS. I can´t dilute my extracts (low target cons). Any problems forseen in SDS-PAGE running? Suggestions how to proceed?

#2 allynspear

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Posted 05 October 2011 - 11:44 AM

Whatever you do, don't add SDS to your samples! If you are using standard PBS, it will contain Potassium Chloride and Potassium Phosphate. When you mix Potassium with SDS you get MAJOR precipitation of Potassium-DS (KDS) and your protein (same thing happens in minipreps after neutralization step). In 1X PBS, the potassium concentration might be low enough to get away with it, but in 10X PBS I'm pretty sure you will lose all your sample.

The only solution I can think of is to dialyze your samples against some other buffer without Potassium to get rid of it. Otherwise you may need to start over. In that case, it may not hurt to run the gel and see how it looks, just in case your samples are hard to prepare.

If you are really lucky, you might be using a PBS that is only Sodium Chloride and Sodium Phosphate (not real PBS), in which case you should be okay.

Best of Luck.

#3 Mirandish

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Posted 05 October 2011 - 11:44 PM

Thanks - highly appreciated

#4 mdfenko

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Posted 13 October 2011 - 05:39 AM

it still won't be okay. that much salt in the sample will cause problems with electrophoresis.

you can "drop" dialyze some sample for electrophoresis.
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