Visualization of proteins in gels

As various proteins are not striate visible on gels to the naked eye, a technique has to be employed in order to visualize them following electrophoresis.  The most generally used protein stain is the dye Coomassie brilliant blue. In an acidic alcoholic solution of the dye following electrophoresis the gel holding the separated proteins is immersed. This denatures the proteins fixes them in the gel so in which they do not wash out and allows the dye to tie to them. After washing away excess dye the proteins are visible as discrete blue bands. As little as 0.1–1.0 μg of a protein in a gel can be visualized by using Coomassie brilliant blue. A more sensitive common protein stain includes soaking the gel in a silver salt solution. Moreover, this method is rather harder to apply. If the protein  sample  is radioactive  the proteins  can  be visualized  indirectly  through overlaying   the  gel  with  a  sheet  of  X-ray  film.   The radiation emitted will occurs a darkening of the film with time hours to weeks depending on the radioactivity of the sample proteins. Upon development of the film the resulting autoradiograph will have darkened field corresponding to the positions of the radiolabeled proteins.

One more  way  of  visualizing  the  protein  of  interest  is  to  use  an  antibody beside  the protein  in an immunoblot Western  blot. For this method, the proteins have to be transferred out of the gel on to a sheet of nylon membrane or nitrocellulose.  This  is accomplished  through  over- laying  the  gel  with  the  blotting    and nitrocellulose  the  protein  on  to  it  through applying  an electric  current.  The nitrocellulose then has a precise image of the pattern of proteins which was in the gel. On the nitrocellulose the excess binding sites are then blocked with a nonspecific protein solution like as milk powder, previously placing the nitrocellulose in a solution containing the antibody which recognizes the protein of interest (the primary antibody). After erasing excess unbound antibody the primary antibody which is now specifically bound to the protein of interest is detected with either a radiolabeled, enzymecoupled or fiuorescent secondary antibody. At last, the secondary  antibody is detected either through placing the nitrocellulose against a sheet of X-ray film if a radiolabeled secondary  antibody  has  been  used,  through  using  a  fiuorescence   detector  or  through adding  to the  nitrocellulose  a solution  of a substrate  which  is transformed  into  a colored insoluble  product via the enzyme which is coupled to the secondary  anti- body.