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screening Protein Expression

Protein expression

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#1 ahmadfathi

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Posted 17 July 2013 - 11:23 PM

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?

#2 labtastic

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Posted 18 July 2013 - 05:56 AM

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.





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