Question

After running your purified DHFR fusion protein+Laemmli 40uL sample, along with a protein ladder (Kaleidoscope standard you used in lab), you transfer protein on the gel to a nitrocellulose filter for Western Blot analysis.

*THE FUSION PROTEIN WAS IDENTIFIED TO BE HIS

a) Which of the following primary antibodies could you use to detect your fusion protein based on the fusion protein identified and process described above: Anti-Myc, Anti-FLAG, Anti-GST, Anti-His, Anti-DHFR, Anti-Tubulin? List all that apply:

b) List how many bands of what size (kDa) are expected from analysis of your prepared fusion protein sample. (2pt) PLEASE EXPLAIN

c) Does it matter which of the antibodies you use from those identified in (a.)? Briefly explain the rationale for your answer.

Answer 1

A. Since its a His tag DHFR protein so for identification through western blotting you need to use Anti-His as well as Anti-DHFR to observe on the membrane, So primary antibodies to be used for this Anti-His & Anti-DHFR, please note that it is a recombinant product where DHFR is human protein expressed in E.coli and His tagged and HRP conjugate can be used as secondary antibody through ECL system.

B. You may expect band size of this fused protein is ~24 Kda on nitrocellulose membrane (exact molecular weight of this Tag protein is 23.6 kda), use ladder for determination, on the SDS PAGE the actual band may vary than the expected.

C. Out target is to see the presence of DHFR as it is the target protein and His is acting as a tag or probe and if its fused then either primary antibody can be used anti-His or anti-DHFR but if His tag is cleaved then it is pertinent to use anti-DHFR antibody. In the given scenario His tag is fused with target protein (DHFR).