Describe a staining method (including reagents and procedure) that could be used
ID: 173205 • Letter: D
Question
Describe a staining method (including reagents and procedure) that could be used to visualize the proteins on the membrane prior to immunostaining. Why is this step necessary? A student was two SDS-PAGE gels. One of the gels was stained with Coomassie blue, and the other was electroblotted. The membrane is a replica of the gel's protein pattern, and is subsequently stained with an antibody. Two 1 degree antibodies (A & B) were purchased from different companies for protein X used to probe membrane, and the results obtained companies are shown below (Figures 1-3). Coomassie blue stained gel showing the protein pattern. Lane 1, pre-stained molecular weight marker; lane 2, tissue extract; lane 3, protein purified by gel filtration; and lane 4, affinity purified protein X. Western blot of Figure 1 probed with 1 degree antibody AExplanation / Answer
1. There are various staining methods for protein staining at non- specific manner; it stains all the proteins in the membrane whereas immune staining is specific to the desired protein using specific labelled antibodies. Following are method of staining:
Coomassie R-250:
a) Transfer your protein on the membrane (example: PDVF membrane)
b) Soak the PVDF membrane in methanol
c) Stain PVDF membrane with 0.1% Coomassie reagent in 40% methanol for 15-20 seconds. Staining for longer period is avoided because it gives dark background which interferes in protein detection.
d) Destain the membrane with 50% methanol
e) Rinse the membrane with deionised water
f) Observe the required protein with the help of ladder
Ponceau S:
a. Transfer your protein on the membrane (example: PDVF membrane)
b. Stain the PVDF membrane for 1-3 minutes with 0.25% Ponceau S in 1% Acetic acid until proteins bands start appearing destain in D.I. water
c. Rinse the membrane with deionised water
d. Observe the protein bans with the ladder.
2. Figure 1:
As mentioned earlier, Coomassie is a dye which stains all the proteins and is non-specific. That’s why it is showing all the proteins bands. First lane contains ladder showing bands of different molecular weight. 2nd lane is showing all the proteins taken out from the call. 3rd lane is showing the proteins separated by gel filtration chromatography which separates proteins on the bases of molecular protein. In 4th lane protein is purified with affinity chromatography, it’s a very specific method based molecule’s properties (enzyme-substrate, antigen- antibody and receptor-ligand interactions).
Figure:2
This is showing the immune-blotting in which specific labelled antibodies are used to detect the target protein. Here single type of antibody is used for labelling and that’s why showing a single band with same molecular weight.