2 replies to this topic

[Grant1derlin]

Hello,

 

Me and my PI are doing a simple protein gel prior to a normal western to see if our protein was successfully expressed in BL21. The following protocol is a rough outline of what we hope to use on 10 mL volumes spun down to pellet before committing the time and reagents to a proper purification and western blot to see if we have protein. I was hoping some people could look it over before hand and over any advice/their opinions. 

 

1. Resuspend pellet in an 8M urea solution (with protease inhibitor) and let sit on ice for 30 minutes

2. Spin down 5 minutes at 10k rpm

3. Discard supernatant

4. resuspend in 8M urea solution and add 20uL Ni resin (transfer 1mL to eppendorf tubes) 

5. Let sit for 30 minutes on ice

6. Spin down and remove supernatant 

7. Add 8M urea and wash 2x

8. load 20uL beads + loading buffer into gel and run.

 

Note: we are just hoping that this will make it easier to see out target bands rather than simply lysing the pellets with 8M urea solution and running it on a gel as there might be too much background from the bacterial proteins.  

[mdfenko]

are you saying that your expressed protein is not soluble in urea?

 

urea denatures (often reversibly) and solubilizes proteins. if you discard the supernate then you may be discarding your protein of interest.

[labtastic]

Why use urea in the first place? Do you know it expresses in inclusion bodies?

 

If it expresses in inclusion bodies, isolate the inclusion bodies and you'll have ~50% pure protein right off the bat so background issues will be minimal. If it is soluble, then don't use urea as you'll solubilize more protein than you would if you just took the soluble fraction, increasing your background.

 

Nevertheless, I don't think it is necessary to do both nickel purification AND a western blot. Granted you say you want to avoid as much background as possible, but in principle you shouldn't need the double specificity (nickel + western) for a simple experiment like testing whether expression worked or not. I would do a single method conferring specificity (either nickel or western). If it were me I would do the nickel purification then stain with coomassie (and if coomassie isn't sensitive enough do a TCA/acetone precipitation before hand). Even if there is a little background, do everything side-by-side with cells that did not express your protein of interest and you will easily see what bands are background and what are signal. 

Edited by labtastic, 29 February 2016 - 09:33 AM.