Problem with protein transfer in non-reducing conditions - (Oct/03/2006 )
Hi all!
I ran the same proteins in both reducing and non-reducing conditions (side-by-side) using 10% Bis-Tris SDS-PAGE and MOPS. I was able to detect bands in the reducing conditions, however not in the non-reducing condition. The antibody should work in both non-reducing and reducing (according to manufacturer) and the ladder was successfully transfered to the nitrocellulose membrane in both cases as well. Next, I used amido black to stain the nitrocellulose membrane to detect all protein...but other than the ladder, there was no protein on the non-reduced blot (in contrast to protein bands stained on the reduced blot).
I appreciate any help or suggestions so that I may successfully transfer protein in non-reducing conditions 
-lil-
From my experience, a ladder transfer is not always the best indication that your protein is there. Could it be that your protein went through the membrane, ie that you transfered for too long?
-tap14-
QUOTE (tap14 @ Oct 4 2006, 09:59 AM)
From my experience, a ladder transfer is not always the best indication that your protein is there. Could it be that your protein went through the membrane, ie that you transfered for too long?
Thank you for the reply. That is definitely a possibility.
For several years, I have successfully transferred for 1 hour using 30V (1.5 hours if running 2 gels simultaneously).
Is transfer rate different in non-reducing conditions?
I will run the experiment again and stain the gel for protein (which I should have done before!) to make sure that it has transferred.
-lil-
QUOTE (lil @ Oct 4 2006, 06:25 AM)
Hi all!
I ran the same proteins in both reducing and non-reducing conditions (side-by-side) using 10% Bis-Tris SDS-PAGE and MOPS. I was able to detect bands in the reducing conditions, however not in the non-reducing condition. The antibody should work in both non-reducing and reducing (according to manufacturer) and the ladder was successfully transfered to the nitrocellulose membrane in both cases as well. Next, I used amido black to stain the nitrocellulose membrane to detect all protein...but other than the ladder, there was no protein on the non-reduced blot (in contrast to protein bands stained on the reduced blot).
I appreciate any help or suggestions so that I may successfully transfer protein in non-reducing conditions
I ran the same proteins in both reducing and non-reducing conditions (side-by-side) using 10% Bis-Tris SDS-PAGE and MOPS. I was able to detect bands in the reducing conditions, however not in the non-reducing condition. The antibody should work in both non-reducing and reducing (according to manufacturer) and the ladder was successfully transfered to the nitrocellulose membrane in both cases as well. Next, I used amido black to stain the nitrocellulose membrane to detect all protein...but other than the ladder, there was no protein on the non-reduced blot (in contrast to protein bands stained on the reduced blot).
I appreciate any help or suggestions so that I may successfully transfer protein in non-reducing conditions
what is your reducing agent? you only mention SDS which means denaturation+ reduction vs non-denaturation+ non-reducction? SDS is not a reducing agent
there should some difference in running and transfer rates as non-reduced proteins have some third structure appearance and run less "elegant" through acrylamide gel than third-structure-denaturated protein which is also true for blotting; so if you blot your gels in parallel the reduced one should faster blot than non-reduced
-The Bearer-
QUOTE (kosmodrom @ Oct 4 2006, 04:22 PM)
QUOTE (lil @ Oct 4 2006, 06:25 AM)
Hi all!
I ran the same proteins in both reducing and non-reducing conditions (side-by-side) using 10% Bis-Tris SDS-PAGE and MOPS. I was able to detect bands in the reducing conditions, however not in the non-reducing condition. The antibody should work in both non-reducing and reducing (according to manufacturer) and the ladder was successfully transfered to the nitrocellulose membrane in both cases as well. Next, I used amido black to stain the nitrocellulose membrane to detect all protein...but other than the ladder, there was no protein on the non-reduced blot (in contrast to protein bands stained on the reduced blot).
I appreciate any help or suggestions so that I may successfully transfer protein in non-reducing conditions
what is your reducing agent? you only mention SDS which means denaturation+ reduction vs non-denaturation+ non-reducction? SDS is not a reducing agent
there should some difference in running and transfer rates as non-reduced proteins have some third structure appearance and run less "elegant" through acrylamide gel than third-structure-denaturated protein which is also true for blotting; so if you blot your gels in parallel the reduced one should faster blot than non-reduced
I use the NuPAGE system by Invitrogen -
In reducing conditions:
the reducing agent supplied contains 2,3-Butanediol, 1,4-
dimercapto-, ( theta, theta)-(+/-)- and is added to each sample along with the LDS sample buffer. Antioxidant (N,N-Dimethylformamide;Sodium bisulfite) is then added to the inner chamber of the running buffer (MOPS SDS running buffer).
Non-reducing conditions - I don't add the reducing agent to the protein sample or the antioxidant to the inner chamber
Yes, I agree that the transfer rates are different. I will run another one and experiment a bit with the transfer times.
Thank you for your comments.
-lil-