Western blot of phosphoproteins - (Jul/21/2005 )

I got some problems when doing western blot of phosphoproteins. Briefly, the proteins were separated from WCLs on Qiagen columns. Eluates were concentrated on Amicon 10kD, cleaned with 2D Clean-Up kit (Amersham), redissolved in Destreak rehydration buffer (Amersham), and subjected to 2D mini gel-electrophoresis. I've loaded 50 mcg of total protein on the gel to be silver stained and 10 mcg- on the gel for transfer and blotting. Silver stained gel showed good resolution and amount of protein corresponding to 50 mcg (from my experience). Another gel was transferred on Hi-Bond PVDF membrane (I used this kind of membrane s for a while and it was always ok). All prestained standards were completely transferred (Except for the partial transfer of 220 kD standard). I did not use SDS in the transfer buffer. But western blot with anti-pS/pT revealed very few, very weak signals. Even more, the antibodies labeled protein standards.
The proteins are presumably mostly phosphorylated ones. So, we suppose to get a lot of strong signals. What can be the problem here? If anybody has an experience with western blot of phosphoproteins?

hi

i think you are overloading your western. i usually load 200ng. if the signal is too great, the blot will be white. maybe that's why you are not seeing anything. did you put a positive control on the blot?

hope this helps! good luck!

I got some problems when doing western blot of phosphoproteins. Briefly, the proteins were separated from WCLs on Qiagen columns. Eluates were concentrated on Amicon 10kD, cleaned with 2D Clean-Up kit (Amersham), redissolved in Destreak rehydration buffer (Amersham), and subjected to 2D mini gel-electrophoresis. I've loaded 50 mcg of total protein on the gel to be silver stained and 10 mcg- on the gel for transfer and blotting. Silver stained gel showed good resolution and amount of protein corresponding to 50 mcg (from my experience). Another gel was transferred on Hi-Bond PVDF membrane (I used this kind of membrane s for a while and it was always ok). All prestained standards were completely transferred (Except for the partial transfer of 220 kD standard). I did not use SDS in the transfer buffer. But western blot with anti-pS/pT revealed very few, very weak signals. Even more, the antibodies labeled protein standards.
The proteins are presumably mostly phosphorylated ones. So, we suppose to get a lot of strong signals. What can be the problem here? If anybody has an experience with western blot of phosphoproteins?

I got some problems when doing western blot of phosphoproteins. Briefly, the proteins were separated from WCLs on Qiagen columns. Eluates were concentrated on Amicon 10kD, cleaned with 2D Clean-Up kit (Amersham), redissolved in Destreak rehydration buffer (Amersham), and subjected to 2D mini gel-electrophoresis. I've loaded 50 mcg of total protein on the gel to be silver stained and 10 mcg- on the gel for transfer and blotting. Silver stained gel showed good resolution and amount of protein corresponding to 50 mcg (from my experience). Another gel was transferred on Hi-Bond PVDF membrane (I used this kind of membrane s for a while and it was always ok). All prestained standards were completely transferred (Except for the partial transfer of 220 kD standard). I did not use SDS in the transfer buffer. But western blot with anti-pS/pT revealed very few, very weak signals. Even more, the antibodies labeled protein standards.
The proteins are presumably mostly phosphorylated ones. So, we suppose to get a lot of strong signals. What can be the problem here? If anybody has an experience with western blot of phosphoproteins?