Intern Needing Help - (Dec/28/2011 )
I'm a research intern at MD Anderson Cancer Center looking for tips to make my blots look "right." My problem that I've been facing is that I keep getting these weird lines. I defrost and use WCL with inhibitors on application. I will also add that I accidentally boiled these samples longer than usual (99C for 20 minutes). I usually do 10 minutes.
The protein of interest in my project is 98 kDa.
Check the salt concentration of your buffers, particularly the lysis buffer.
over boiling can cause protein loss due to aggregation. 5 minutes should be more than sufficient.
you can try ~65C for 10-20 minutes if you want to avoid aggregation during long incubations.