Purification Rookie - advice appreciated - (Feb/01/2008 )

Hi everyone, I'm looking for some feed back on the following protocol for purifying proteins. Should this technically work? I've tried running through this a few times and keep getting inconsitent results (sometimes protein, sometimes no protein, some times very dilute protein......) I'm not really sure where I'm going wrong.

I think I've always made up new buffers before starting, so I dont think using 'old buffers' is an issue.

Also, do I need to establish whether my recombinant protein is in the soluble or insoluble fractions before even beginning all of this work? How do I do that?

Sorry for the long post! Here's the protocol I'm wondering about:

His(6) protein purification

1. wash pellet in PBS
2. lyse in BUFFER A PLUS PROTEASE INHIBITORS (use about 30 mL of buffer for each pellet  each pellet comes from a 1 L overnight culture of Magic Media (Invitrogen)) – sonicate 4x15 seconds, all on ice. Use setting “60” on sonicator
3. centrifuge (25 min, “10” setting on large rotor centrifuge) – while spinning, wash a Ni2+-IDA (GE [amersham]) column in BUFFER A (10 mL), followed by equilibration in BUFFER B (10 mL)
4. load cleared sample onto the equilibrated column – collect 8 uL of the flowthrough and place in a separate tube with 2 uL of 5X SDS loading buffer.
5. wash with BUFFER B (15 column volumes [15 mL for the HisGravitrap kit columns]) – collect 8 uL of the wash fraction and place in a separate tube with 2 uL of 5X SDS loading buffer.
6. elute with BUFFER C, 2x3mL, collect 6 fractions (1 mL each) - collect 20 uL from each elution fraction and place in a separate tube with 5 uL of 5X SDS loading buffer.
7. Verify fractions containing your purified protein via SDS-PAGE.
8. Desalt samples: equilibrate a PD-10 column (GE[amersham]) with 25 mL of DMEM. Apply the fractions identified in Step 7 to the equilibrated column. Elute with 6 mL of DMEM without phenol red, collect 1 mL fractions, and analyze via SDS-PAGE as in step 6.
9. Pool desalted fractions containing your protein and add ~10% glycerol. Do a protein determination for a small volume (tend to be very concentrated – maybe use 1 or 2 uL). Store at -70 degrees until ready to use.


STOCK SOLUTIONS:
5M Imidazole – 17.02 g in 50 mL
2.5M NaCl – 29.22 g in 200 mL
2M Tris-HCl pH 7.9 – 63.04 g in 200 mL (pH to 7.9)


BUFFER A
6M Urea
5 mM Imidazole
500 mM NaCl
20 mM Tris HCl pH 7.9
Protease inhibitors

BUFFER B
6M Urea
20 mM Imidazole
500 mM NaCl
20 mM Tris HCl pH 7.9

BUFFER C
6M Urea
1 M Imidazole
500 mM NaCl
20 mM Tris HCl pH 7.9


Notes: TAT-GFP, TAT-RHOA, TAT-GFP-RHOA did not bind well to the column when equilibrated with 80 mM imidazole in Buffer B; use 20 mM instead.

Can you see your protein in any of the samples you took before elutions? I track my protein by taking samples every time something happens to the protein, like during sonication, spin down, wash, etc to make sure it doesn't run off somewhere. You said you get inconsistent results.. are you using the same protein/cells?
The buffers you are using contain urea as a denaturing reagent so it shouldn't matter if your protein is soluble or not (I personally use two different protocols depending on solubility though). Your protocol looks sound to me. I find that most of my problems seem to occur around induction time (wrong OD, bad IPTG), before I even start prepping my pellet.

To figure out if your protein is soluble or insoluble, don't use urea in your lysis buffer. After sonication and centrifugation, a soluble protein will be in the supernatant. Insoluble proteins will be in the pellet. To go after the insoluble pool, resuspend the pellet with buffer containing 6M guanidine or 6M urea.

Oh yeah, two other things that I do with my preps.. I lyse my cells with lysozyme before sonication. After I get my lysate, I add beads directly to the supe and incubate for 1-2hrs. Then just load the whole thing onto my column. If your lysate is rather viscous, it helps to spin down your beads (save a sample of supe) and resuspend in wash buffer, then load.

I have been taking samples everytime I manipulate the cells in some way, but I was having difficulty tracking things with westerns (I had problems with my gels/buffers at one point, so unfortunately this problem occured at the same time I was trying to purify things - made it very difficult!). I have in fact detected my protein of interest on a couple of different occasions through westerns, after purification and even after desalting, but on subsequent attempts I was unable to get a signal on western. I will try again though - and hopefully be more careful next time.

What beads are you referring to, after the addition of lysozyme? Ni-NTA beads? or just something to clear away bacterial proteins?

come to think of it, I've never used lysozyme either, what does that do? is sonication not enough to lyse bacterial cells?

EDIT - although I've never used it - it turns out we do have some in the lab, from Sigma. The offer a sample protocol for use of the product in the datasheet (http://www.sigmaaldrich.com/sigma/datasheet/l6876dat.pdf) but it seems this is directed toward isolating plasmid DNA, is this what I should be doing with a BL21 strain from which I'm trying to recover my expressed protein? Do I just add the lysozyme to my 'lysis buffer' (containing Urea?)?

Thanks for the help,

H