Protein Aggregation, Sample Boiling, help!
Posted 20 May 2011 - 07:11 AM
Posted 30 July 2011 - 08:35 PM
Try lowering the concentration of the protein in the lysate - use more lysis buffer or fewer cells.
Posted 03 August 2011 - 10:12 PM
I believe I'm having a major protein aggregation issue with my whole cell lysates, but I can't pinpoint the cause. The thick prominent band in my 10% blot (examples pictured below) is a non-specific band. I am looking for pSmad2 which is around 60 kD, so this thick band runs right through it. I boil my samples for 5 min at 100C immediately after lysis and before loading into the gel. My DTT concentration is 150 mM. I have tried centrifuging before loading, extensive sample dilutions, and I've had no sign of improvement. What else can I do to break up this band?
I have the exact same problem, have you been able to figure out how to fix it!!
Posted 04 August 2011 - 06:31 AM
is there residual cell culture medium with the cells when you lyse with sample buffer? you may be seeing albumin from the serum in the cell culture medium (it can show a depressed molecular weight when overloaded).
if so then you may want to wash your cells with serum-free medium a couple of times prior to lysis
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