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Boiling, protein aggregation......reversal?? (western)


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3 replies to this topic

#1 Sora

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Posted 06 May 2011 - 02:27 AM

Hi,
I wanted to run some important samples on SDS PAGE today but I made this really stupid mistake of boiling my protein samples for like 20mins at 95C instead of 5mins :( (thats what happens when you're working under pressure). Is there anyway I can reverse the damage caused by over-boiling. Right now, theyre sitting on ice. Im looking for a proteins at about 20kDa. Just to let you know, my sample buffer had both SDS and BME!

Thanks

#2 BioMiha

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Posted 06 May 2011 - 04:59 AM

Just run the samples. What damage can be caused by overboiling?

#3 uttess

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Posted 06 May 2011 - 07:33 AM

i think your sample got denatured..i did the same mistake..my protein is 28 kDa....i could not see the band, i could nt see actin (the loading control too)

#4 vegeta

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Posted 06 May 2011 - 09:48 AM

The problem derives from over-denaturation which may cause the proteins to form hydrophobic interactions with each other. I really don't know how to reverse this. Try adding urea or glycerol??




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