WWA
Nickel Purification and Coupling to Affigel (Beth 12/14/00)
1) Thaw the pellets at 37°C with 175-200mLs of extraction buffer (containing
PIs) per 18 Liters of expressed cells, add PMSF to 1mM just prior to lysis and
no DTT or BME! Check the thickness so
that lysis occurs readily- too little volume and you will not get complete
lysis.
2) Combine all thawed pellets in one beaker and microfluidize with 3 complete passes through the microfluidizer.
3) Put into SS34 tubes and spin at 18,000rpm for at least 40 minutes. Save the supernatant in a beaker on ice and keep the pellets (just in case). Meanwhile, equilibrate the Nickel resin in1X extraction buffer at least 5 times in the Falcon Tube (fill up to 50mLs and decant the wash and repeat 5 times), you can do this quickly by spinning the resin in the clinical centrifuge. Also pour 2-4 SDS Page gels so that you can check the eluates.
4) Use around 20-25mLs of Nickel resin that has been equilibrated in 1X extraction buffer. Combine the supernatant with the Nickel resin in a beaker and stir at 4°C for 20 minutes, Pour into a column and collect and save the flow-through just in case.
5) Wash column in 200mLs of binding buffer, if you use wash buffer with imidazole the WWA tends to come off. Save the wash (just in case).
6) Elute the column using 100-250 mls of 1X elution buffer and collect 10mL fractions in (at least 10 fractions). Take 20 microliters of each mixed fraction and make a gel sample. Run all the eluates on a gel with a protein ladder. On other gels run samples of the FT and wash and load fractions. Keep the eluates on ice or in the cold room until you have determined which fractions to combine. For some reason, the His-NWASP construct purifies in the same fractions as a yellowish colored compound- so if you see this, chances are that the WWA is in this fraction.
7) Combine the fractions and dialyze overnight in 10mM Hepes, pH 7.2 + 100mM NaCL. Do not add DTT or BME since a few times with certain resin lots the dialysate turns brown when you add DTT (I think the Ni is leaching off the column)!
8) The next day change the dialysis buffer one time in the morning. A typical yield will give a lot of protein. Usually after looking at a gel, I use 2mLs per 2mL of affigel 10 resin. I aliquot and freeze the rest. Lately we have gel filtered the NWASP after this Ni- purification step. Then you can dialyze it back into the buffer in step 7) and freeze it in aliquots. This buffer is one that JZ determined empirically couples most efficiently to Affigel 10.
NOTE: SAVE all of the column fractions, a Load sample, Flow through and wash samples.
Do NOT use Affigel 10 that is over 6 months old- parafilm the vial after every use.
EXTRACTION Buffer:
10mM Tris
11.6% Sucrose
1mM EGTA
1X PI cocktail
1mM PMSF
0.1mM Benzamadine
8X Binding buffer:
40mM (4mL of 2M) Imidazole
(160mL of 5M) NaCl
160mM (32 mL of 1M) Tris, pH 7.9
Water to 200mL
27.23g Imidazole (4M)
40mL 5M NaCl or 11.69g NaCl (2M)
8mL 1M Tris-HCL, pH 7.9 (80mM)
Water to 100mLs