Boiling, protein aggregation......reversal?? (western) - (May/06/2011 )
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!
Just run the samples. What damage can be caused by overboiling?
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)
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??