Preparing SDS Sample for Soluble/Insoluble Fractions - Really confused as to what the best methods to make these fractions is (Mar/16/2010 )

Hey guys. this is my second post on this forum. First post was answered well, so I thought I would give this a go.

I want to see my pilot expression levels from BL21*(DE3) strain on an Nupage Bis-Tris 4-12% gel with MES running buffer and LDS Sample Buffer(4X).

I have my pellets from 500 microlitre cultures taken at different time points after induction with IPTG. I want to load the soluble and insoluble fractions of all these. time points on my gel.

This is what I did :

1) Used Bugbuster protocol to get my supernatant which has my soluble fraction.
2) For my insoluble pellet, what is the best solution to use to dissolve the insoluble proteins? Some people say just add SDS sample buffer to the pellet and boil. How much of the 4X LDS sample buffer do I add?
3) Some people say redissolve the pellet in 8M Urea, to solublize the insoluble proteins, THEN mix with sample buffer and boil? (But i also heard this makes problems on the gel, is this true?)

What is the best protocol for making the insoluble fraction for loading onto the gel ?

Cheers

for the insoluble protein you can use sample buffer and boil. 4x sample buffer should be diluted to 1x (after solubilizing the pellet). don't boil too long, you may cause the protein to aggregate despite the sds and 2-me (or dtt). you can heat at 65C for extended periods. the pellet should not be so big that you can't get it into solution reasonably quickly. too much protein will also cause overloading and streaking (if not completely solubilized).

you can solubilize with urea but do not heat it. urea will decompose and carbamylate your proteins.

Cheers for the advice! Do I dilute it with distilled water or do I have to use some other buffer?

The pellets that I got from my pilot expression were stored in -20 degree. I took them out a week ago and bugbustered them, and got my supernatant out and put it into another tube. The insoluble fraction is still in pelleted form, and I havent added sample buffer+reducing agent yet.

However, I left the tube outside after I lysed them(i did add protease inhibtor cocktail though) and forgot about it till today. Does this oversight mean any protein in my soluble fraction/insoluble fraction is gone?

Do I have to repeat my pilot expression?

Sharky on Mar 19 2010, 08:58 AM said:

I am interested too,

how the specificity of the insoluble fraction? Does it exist any protein which are only insoluble?

Sorry to revive an old post, but this applies to a problem I'm having.

I'm overexpressing p53 with BL21(DE3) E. coli cells, and when I lyse and spin my protein is in the insoluble fraction. I've resuspended the pellet in 50 mM Tris-HCl, pH 8.0, added 0.5 mL Trition X-100, and repeated. Basically, I've performed this wash step 3 times (as mentioned in another group's pub, who were also expressing p53 with the same vector). And now it's at this point where I'm having difficulty.

My results correlate almost exactly to that of the other group, the only problem is in the literature they say after washing with Triton X-100 three times they got a "firm" pellet. Well, "firm" inadequately describes my pellet, that sucker is stuck. The other group solubilised their pellet with 5 M guanidine-HCl, 50 mM Tris-HCl, 0.005 % Tween 80, pH 8.0 at 4 C for 5 h with rocking. Well, I tried 8 M urea, 50 mM Tris-HCl, Tween 20, pH 8.0 at 4 C with rocking and after 3 h the pellet looked the same as it did when I took it out of the centrifuge.

I think I'll try their protocol (which I avoided and opted for 8 M urea instead because I'm worried about the guanidine-HCl degrading too quickly). However, I would like to use the same pellet I've been working with, the one in urea. So far as I can tell, the pellet hasn't decreased in size, so I imagine my protein should still be in there. Also, I believe I added too much Tween, because after adding it I let it rock in the 4 C for 3 h, and when I returned the pellet was still intact and the buffer had become white, loosely gel-like, and viscous in consistency.

Any suggestions? Can I decant off the urea/tween jello and use the same pellet? Also, any ideas as to how I could dissolve this pellet back into solution so I can run it on a column?

Thanks!

well you can heat at 65C for expanded periods. the pellet should not be so large-scale that you can't get it into answer sensibly quickly. too much protein will furthermore origin overloading and streaking..

ignore shane, he's just translating and retranslating previous responses and adding a spam link.

you can try with the same pellet but save and analyze the solution that gelled. you may have solubilized some protein or gelled the tween or both.

why did you switch tweens? tween 80 and tween 20 are not really interchangeable.

what we do is to solublize the inclusion bodies in 400 µl of a buffer containing 8M urea (dissolved in 100mM Tris/HCl pH 8,2 + 100 mM ß-mercaptoethanol) for 0.5h at RT at a shaker and load this directly on the PAGE gel ...this works fine for us!

Regards,
p