I already check protein expression level on SDS-PAGE gel.
For checking I already used CBB stainning.
But I'm not sure it was my target protein or not.
So on this gel, I want to do transfer to membrane using same gel.
Is that possible to detect band after coommasie stainning?
Or should I have to do destaining step?
While I have never personally tried this, I would suppose that coomassie will unspecifically stain and block your blot membrane, making it inaccessible for your actual protein of interest and therefore screwing up your western blot. Apart from that, coomassie interacts with the protein carboxyl and amino groups, which can make them inaccessible for a detection antibody.
However, you can skip the coomassie step on the SDS gel level, and just go for the procedure: blot - block (2-5% milk) - immunostain - coomassie directly on the PVDF membrane (careful: do not use nitrocellulose!). This serves as nice loading control and is generally cheaper than Ponceau S. For more information on this, read here:
Welinder, Charlotte; Ekblad, Lars (2011-03-04). "Coomassie Staining as Loading Control in Western Blot Analysis". Journal of Proteome Research. 10 (3): 1416–1419. doi:10.1021/pr1011476. ISSN 1535-3893.
Which Coomassie reagent did you use? If you stained the gel in 40 % MeOH 10 % Acetic acid then the transfer will be fairly inefficient. You will observe better efficiency if you stained with the colloidal form of Coomassie (G-250) with lower concentrations of acid/alcohol.
Better still, re-run your samples and transfer onto PVDF/Nitro membrane. Stain the membrane in Ponceau-S then partially destain (in plain water) to visualise the proteins and note the marker/ladder position (or use coloured markers). Take a photo then fully destain the membrane and procede with the immunodetection. You could also run a duplicate gel and stain that in coomassie blue for comparison.
A method for the imumunodetection of proteins after electro-blotting onto a nitrocellulose membrane has been published . After fixing and staining the gel with Coomassie the gel is washed with water and then washed in a buffer containing SDS. The Coomassie pattern appears on the membrane , It is a little more diffuse and fainter the the original gel patter but recognisable. Positive immunodetection is detected easily by a change in colour of the band ( or spot for 2D-gels) from blue to brown. Imaging through a blue filter will show only the brown bands. It is important to use the correct buffer and SDS concentration for washing the gel before the blotting. The time for this washing is also critical: too short a time and the proteins will not leave the gel efficiently and too long and they will be washed out of the gel and in to the buffer. The optimum conditions are described in the paper.
Reference.:
Jackson, P. & Thompson, R.J. (1984). The immunodetection of brain proteins blotted onto nitrocellulose from fixed and stained two-dimensional polyacrylamide gels. Electrophoresis 5, 35-42.
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