Help troubleshoot SDS gel - smear down well - (May/01/2008 )
Hello, I can use some help.
I am running seed microsomes on a pre-cast invitrogen gel:
NuPAGE 10% Bis-Tris Gel. 1.0mm wells.
Running buffer is 1X MOPS SDS
100V about 1 1/2 hr run.
Stained with Commassie blue about 2hrs. (40% methanol. 10% acetic acid, 0.2% commasie blue)
De-stain with 40%methanol, 10% acetic acid.
I am seeing smears all the way down the well. I have tried adding different amount of protein. From 20ug to 40ug and I get the smears you are seeing on this gel. The amount of protein on this is 25ug per lane. I have not run less than 20ug since it start to look faint and I can't get good resolution around 50kD (size of desired protein in study).
Has anyone had this happen?
a couple of things:
salts in the sample buffer may be causing some of the distortion. dialyze salts away.
incomplete denaturing of the protein with sds and mercaptoethanol (or dtt). you may be smearing aggregates. do you boil your samples? how long? you may want to reduce the temperature to ~65C and incubate for 10 minutes.
clarify your sample prior to loading the gel and avoid picking up the pellet when loading the gel.
Your last lane looks decent - what was different there?