problem in western blotting - (Dec/18/2006 )
I run 2D gels and then transfer the proteins to Nc membrane. I do a transfer in cold at 100mA for 2 1/2 to 3 hours. I find that the colored molecular wt marker has been transferred to the membrane, but after developing the blot, very few spots are lit up. I however know that i must have more no of spots. Is it a problem with my transfer? But if the marker is transferred then why not the proteins. Is it a problem with the amount of protein. I load 25, 50 or 100 ug protein per tube gel. Or is it because i do two transfers at the same time using the biorad transfer cell? (This could be a remote possibility). I have earlier used the same protocol for transfer and developing the blots on 1D gels and it has worked perfectly well. The same antibodies and conditions however fail with 2D. Can anyone help
Mythri
Sorry but I don´t really understand when you say "after developing the blot": do you mean verify transfer with Ponceau or the signal of your antibody? 
Well, I suppose you want to mean Ponceau, so could it be that it isn´t sensitive enough to see all the proteins you expect or you have seen by staining your gel with other more sensitive stainings (silver...)?? 
I´ve used Biorad transfer cells, both wet and semidry, always 2 gels at the same time and never had a problem.
Well, I suppose you want to mean Ponceau, so could it be that it isn´t sensitive enough to see all the proteins you expect or you have seen by staining your gel with other more sensitive stainings (silver...)?? I´ve used Biorad transfer cells, both wet and semidry, always 2 gels at the same time and never had a problem.
Well i should have been more clear. What i mean is the signal to my antibody and not Ponceau staining. Also i know that my primary antibody is very good and there are more proteins that have to be picked up by my antibody, becoz a 1D for the same sample gives an excellent signal and a number of bands
Mythri
OK. I suppose that if you see the MW markers, your proteins will have also been transferred. Although it could be that you need more time to complete the transfer and so get a better signal when developing the blot.
One possibility could be that the bands you saw on your 1D gels may not correspond exactly to the same spots on the 2D gel because of the pHselected for your strip (for example for a 3-10 strip, you won´t see the proteins with pI lower or greater than these values).
Also there´s something I don´t understand (sorry), as far as I know, one antibody is better if recognizes only one protein specifically and doesn´t appear several bands, isn´t it? so what do you mean when you say that your antibody is very good and it gives more than 1 band?
Related with the amount of protein: don´t you see a best signal when you load more protein? Do you see always the same?
One possibility could be that the bands you saw on your 1D gels may not correspond exactly to the same spots on the 2D gel because of the pHselected for your strip (for example for a 3-10 strip, you won´t see the proteins with pI lower or greater than these values).
Also there´s something I don´t understand (sorry), as far as I know, one antibody is better if recognizes only one protein specifically and doesn´t appear several bands, isn´t it? so what do you mean when you say that your antibody is very good and it gives more than 1 band?
Related with the amount of protein: don´t you see a best signal when you load more protein? Do you see always the same?
OK it goes like this. My primary antibody is for a protein nitration, it recognises all nitrated proteins. And since my nitrating agent is going to nitrate several proteins in the cell, i have to get a large no. of spots corresponding to all the nitrated proteins
OK, thanks for the explanation 
I don´t know, but could it be that the some of the procedures (first dimension, buffers, reducing agents or whatever) of the bidimensional electrophoresis affects the nitration of some of your proteins so then they are not well recognized? 
Does any bioforumer have another opinion about this problem? 