Visualization of all protein bands is possible with Coomassie staining. Glycoprotein bands with a known molecular weight can be identified by referencing a Molecular Weight Marker lane on the gel or membrane. Phenomenon such as shift changes due to denaturations or digestions can be documented when comparing treated to non-treated samples.
Fetuin was digested with various glycoprotein specific enzymes, PNGaseF (P), neuraminidase (N), and O-glycanase (O). The reactions were separated on a 7.5% NEXT Gel and stained with Coomassie. The total protein stain of the gel confirms the PNGase F activity due to the protein shift in molecular weight. Neuraminidase and O-glycanase activities were confirmed using lectin-specific Western blots.
Fetuin was digested with either PNGase F or Neuraminidase and separated on a 10% NEXT Gel using a preparative comb. The proteins were transferred to Odyssey® Nitrocellulose, the membrane was stained with Coomassie and bands were detected using an Odyssey Imager. The image shows the protein band shifts to a lower molecular weight after digestion with PNGase F (largest shift) or Neuraminidase (smaller shift). Total protein detection can also be done on the same blot after detection with IRDye® infrared dye probes.