Western Blotting Resolving Problem
Posted 15 January 2012 - 06:56 PM
I have some problem with my recent Western Blotting. I am checking for 180kda protein and so i run 8% gel. But for proper resolving I run the gel at 120V for 3.30 hrs (usual timing 2.30 hrs). I am able to see the band of my interest very clearly but not at 180 kda. Its near 140 kDa. If we overrun the gel is there any chances that our protein moves fastly than the marker. Kindly write your experience on this. Thanks in advance..
Posted 16 January 2012 - 04:31 AM
I have experienced problems with pre-stained markers whereby they do not run to exactly the correct size. When we are sizing a new protein band of interest, we would always use unstained markers which you could stain afterwards by eg. amido black.
The pre-stained markers are not 100% accurate for size, they usually say so.
You do not say which markers you use, but if they are coloured already, try unstained markers alongside for comparison. You can then cut them off the gel and stain separately. Your band size might then be correct.
HOWEVER, in a low % gel like yours, the difference in band sizes might be more then can be explained by just this. Ours would run by about a cm different in a 10% mini-gel.
Hope this helps!
Posted 17 January 2012 - 11:29 AM
Incidentally, I would be running a 6% gel for a 180 kDa protein, you won't get proper resolution with an 8% gel.