Blotting Procedures

Transblotting onto PVDF: PVDF is directly compatible with protein sequencers and binds proteins more tightly than nitrocellulose, thereby giving greater recoveries. Transblotting onto PVDF is carried out overnight according to standard procedures using a tris/glycine/methanol buffer system without SDS. Original gels are stained and returned unless otherwise requested. We will change the blotting procedure to your specifications as necessary.

PVDF Coomassie staining and spot cutouts for sequencing: Coomassie blue staining of PVDF does not interfere with protein sequencing. We will quick-stain the blot with Coomassie blue, cut out the 2-D spots or 1-D bands of interest and either send them off for sequencing or return the cutouts to you in an Eppendorf tube so that you can deal directly with your facility.

Transblotting onto nitrocellulose: Transblotting is carried out overnight according to standard procedures using a tris/glycine/methanol buffer system containing 0.1% SDS, with nitrocellulose of 0.45 micron pore size. Dried unstained blots are returned to the client and original gels are discarded unless additional treatments are requested. We will change the blotting procedure to your specifications as necessary:

India ink staining for blots: Nitrocellulose cannot be Coomassie blue stained or silver stained. India ink is the preferred method to visualize patterns on nitrocellulose blots for purposes of comparisons with immunostained blots.

Colored molecular weight markers for blots: These markers are added during slab gel electrophoresis so that lines (2-D gels) or bands (1-D gels) are visible on both unstained gels and subsequent transblots at molecular weights of 95,500, 55,000, 43,000, 36,000, 29,000, 18,400, and 12,400. Note, however, that the thick blue MW lines across blots may interfere with interpretation of horseradish peroxidase staining results.

Western (immuno-) blotting: This method for immunodetection of specific bands or spots is carried out on PVDF transblots from 1-D or 2-D gels. Generally the client supplies the antibody. Our service includes preliminary dot blotting to determine the appropriate antibody dilution (when antigen is available), gel transblotting onto PVDF, preliminary staining of blot with Coomassie blue and antibody detection using the sensitive ECL method. A control membrane run with gel electrophoresis secondary antibody is included as necessary.