screening Protein Expression - (Jul/18/2013 )

hello, right now i doing trials on protein expression. in this method i tried until harvesting the cell..so there already many chemical i wasted and my supervisor really don't like it. Is there any method to screen the expressed protein before doing the harvest cell protocol?

It depends on how you expressed your protein and how well it expresses.

1. If your protein expresses well enough, you can simply run SDS-solubilized cells out on a gel and look to see if there is a big fat protein band at your expected molecular weight. Run this sample side-by-side with cells that did not express protein.

2. If you have a his-tag on it, then you can lyse cells in SDS, run on SDS-PAGE, and then blot with an anti-his tag antibody.

3. Express your protein with a cleavable c-terminal GFP tag. You can then either measure GFP fluorescence of whole cells and compare fluorescence measurements to a native GFP standard curve, or run SDS-solubilized cells out on SDS-PAGE and image the gel by fluorescence (note: to do the latter experiment, you cannot boil your SDS-PAGE samples prior to running the gel otherwise you will denature the GFP and you won't observe fluorescence).

4. If none of those are options, then just do a crude, small scale his-tag purification (assuming your protein is his-tagged). You can do the whole enrichment with a 5 ml culture, <10ml of the appropriate wash and elution buffers, 1 eppendorf tube, and a table top microcentrifuge, i.e. at almost no cost except your time.