Problem with protein resolving in electrophoresis - (Jul/14/2011 )

Hey Friends

My proteins are not resolving properly...in Gel electrophoresis...In the blots its looking like all the proteins getting stuck at a certain point and not going down below that point......can you guys suggest something what are the different problems that can cause this ....I am using a 8% resolving gel which worked very well earlier for the same protein I am looking for..and I am making sure that all the simple steps of electrophoresis are accurate.........waiting eagerly for response..thanks

G1R2

picture?

have you tried with fresh buffer?

old sds can also cause problems.

too short separation of polypeptides may be caused by salt in the protein sample

Hi this is the blot of my proteins....In this blot I have probed the membrane for all glycosylated proteins....according to my earlier good experiments i expect the proteins between 250 to 75 kD...but you can see that they have not come down at all...all of them got stuck to this same position in my other repeat experiments too...and I have longer exposure of this blot also..there also...nothing is there below this point....

mdfenko on Thu Jul 14 19:03:39 2011 said:

Hi this is the blot of my proteins....In this blot I have probed the membrane for all glycosylated proteins....according to my earlier good experiments i expect the proteins between 250 to 75 kD...but you can see that they have not come down at all...all of them got stuck to this same position in my other repeat experiments too...and I have longer exposure of this blot also..there also...nothing is there below this point....

you may have over boiled the samples and ended up with aggregated proteins. try boiling for 5 minutes or less or heat at 60-70C for 10-20 minutes.

mdfenko on Mon Jul 18 20:09:13 2011 said:

Hi

Thanks a lot for your response.....

But the protein ladder i am using ( Precision Plus Protein Kaleidoscope Standards from Biorad) used to look very good but have started looking like it is not resolving well after the 100 kD band ( not showing up distinct bands for 75 , 50 , 37 kD.... ...I am not boiling the standard then why it is also looking like that.......can you please suggest me something..

How long are you running those gels?

At 8% resolving gel, it shouldn't take that long to run a mini gel, like ~less than 1h at 150v, but if it is a large gel probably longer... like 3 h. What % stacking gel are you using? I saw your WB. Your STD (standard) are running short. You do need to run the gel longer so you can mark below 75kDa. Also, did you try commassie staining to make sure that is proteins are not stuck in the gel or not being transferred well? IF dye front is running all the way (almost running out of the box), your electrophoresis is good.

If you transferred too long --> you will get only top STD since low MW protein markers will blow through (lost) in transfer buffer. You need to focus which protein you want to look into. From 250kDa to 75kDa is little too broad.. You might well as use gradient gel.

Check your transfer time and the gel after proteins separated. If nothing shows up in gel except top proteins on gel after staining, all those proteins went through the gel and got lost causing high MW to hang around still. Fully submerged transfer system works better than semi-transfer.