Question

4. A protein sample contains a single protein made up of two dissimilar polypeptide chains of 25,000 daltons and 45,000 daltons joined together by disulfide bonds. Following SDS-PAGE how many bands do you expect to see in the gel if the sample was heat treated with an incubation buffer a. containing a reducing agent such as dithiothreitol? b. devoid of a reducing agent? Explain your answer. 5. Indicate the function of the following compounds in SDS_PAGE. a. Bromophenol blue b. Bis-acrylamide c. SDS d. Ammonium persulfate e. TEMED f. Coomassie blue

Answer 1

PolyAcrylamideGelElectrophoresis is used in biology to separate nucleic acids and protiens with respect to their electrophoretic mobility. A denaturant is required to remove the highly ordered strucure of these biomolecules. Urea is the common denaturant for ncleic acids whereas SDS is used for protiens. Dithiothreitol (DTT) denatures the proteins by reducing disulfide linkages. This helps to overcome some forms of tertiary protein folding, and breaking up quaternary protein structure. After electrophoresis, the gel may be stained for the visualization of the separated proteins.

a) in presence of reducing agent,the S-S bond breaks to the corresponding thiol. Hence is the above case two bands of 25kDa and 45kDa would appear

b)In the adsence of reducing agent only one band would appear

5.a) Bromophenol blue: This is a tracking dye for the colorless protiens

b) Bis acrylamide: this is the most frequently used cross linking agent for polyacrylamide gels

c) SDS: It is a detergent used to denature native proteins to unfolded, individual polypeptides.

d) Ammonium persulfate: This is a source of free radical in the gel formation process.

e) TEMED: This is used for the stabilization of free radicals to improve polymerization and gel formation

f) Coomassie blue: This is a stain for visualizing the bands.