Question

Which of the following is NOT a consideration for detection of your purified expressed protein in Western blot?

1. The protein must have the His-tag removed before gel electrophoresis

2. The protein must be denatured with SDS, boiling and BME before gel electrophoresis

3. The expressed protein should be purified using an affinity column before gel electrophoresis

4. The expressed protein should be eluted with imidazole which mimics the His before gel electrophoresis

Answer 1

The following is not a consideration for detection of purified expressed protein in western blot is :

THE PROTEIN MUST BE DENATURED WITH SDS, BOILING AND BME BEFORE GEL ELECTROPHORESIS.

As the western blot is a technique of identification of protein from already seperated proteins there is no consideration of step of denaturation of protein using SDS followed by boiling. This causes loss of recovered protein in tge blotting technique.

• Affinity chromatography helps in seperation of antigen antibody complex where the protein was incubated with primary and secondary antibody during the process of technique
• The His tagged protein is to be removed before electrophoresis for qualitative detection of protein which was added at either the N or C terminus of a recombinant protein
• Imidazole is in the purification of His-tagged proteins bound to nickel ions attached to the surface of beads.