3 replies to this topic

[doctorlambchops]

Hello Everyone.

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.

Attached Files

•  Sampe Protein that needs fixing.bmp   960.05K   64 downloads

Edited by doctorlambchops, 28 December 2011 - 09:28 AM.

[doctorlambchops]

The protein of interest in my project is 98 kDa.

[bob1]

Check the salt concentration of your buffers, particularly the lysis buffer.

[mdfenko]

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.