Protocol

Abbreviation

uva_purification_1

Name

typical purification protocol at Wladek Minor group

Laboratory name

University of Virginia

Type

purification

Description

Protein Purification Protocol

BUFFERS

All of these buffers contain 5mM beta-mercaptoethanol(BME) if proteins highly susceptible to oxidation such as Se-Met proteins or proteins with high cysteine levels are to be purified.

Binding/Wash/Elution Buffers (BB/WB/EB):
500mM NaCl
50mM HEPES (substitute Tris in the WB) pH 7.5
5% Glycerol
5/30/250mM Imidazole respectively for BB/WB/EB

Dialysis Buffer:
500mM NaCl
50 mM HEPES pH 7.5
5% Glycerol

Crystallization Buffer:
500mM NaCl
10mM HEPES pH 7.5

Note: All steps are performed at 4 degree (in a cold room or on ice, or both) unless stated otherwise! Samples taken for SDS-PAGE mixed with SDS sample buffer should be stored at -20 degree until the gel is run.

DAY ONE

1.Reserve the sonicator and the centrifuge and turn on the centrifuge to cool it to 4 degree.

2.Thaw cells on the bench, and mix 10uL of cells with 30uL of sample buffer for SDS-PAGE analysis. You may also prepare the columns during the thaw (see step #7).

3.Add protease inhibitors (1mM of PMSF and benzamidine) to the cells. The protease inhibitor c degreektail (PIC) st degreek has 50mM of each st degreek, making it 50x. Thus, to 30mL you would add 600uL of PICs. The PICs are neurotoxins, so make sure to wear gloves and handle them carefully!

4.Sonicate the samples for 8 x 30 seconds with 30 second breaks in between.
With a Branson sonicator, use a 1/2 in tip.

5.Add PICs to the lysate once more to a 1mM final concentration (e.g., 600 uL for 30mL of cells).

6.Spin the lysate at 18,000 rpm at 4 degree for 45min (use the Sorvall centrifuge in the center hallway with the Sorvall SS34 rotor).

7.While the lysate is spinning, prepare the columns as follows:
1.DE52 (Whatman), mix 10g of DE52 in 100mL 2.5M NaCl, add to large column (ID 2.5cm) and equilibrate with 40mL BB (binding buffer).
2.Ni resin (Qiagen), 5mL bed volume, to a 20cm column (ID 1.5cm) equilibrate with 20mL BB.
Place the columns in series to flow the samples by gravity or pump in the cold room.

8.Remove an aliquot from the pellet (a small chunk resuspended in 100uL sample buffer) and supernatant (20uL in 20uL sample buffer) for SDS-PAGE analysis.

9.Apply the supernatant from the spin to the upper DE52 column and allow the eluate to flow directly through the lower Ni column. Save and label this flowthrough for SDS-PAGE analysis. We will save the whole flowthrough in case the protein doesn't bind, but take 20uL of this and put in 20uL of sample buffer for the actual gel.

10.Add 2 x 10mL of BB to the DE52 column and again allow the flowthrough to pass through to the lower column.

11.Retain 10mL of BB in the Ni column and add 100uL of PICs, then allow the remaining flowthrough to pass through.

12.Wash the nickel column with 100mL WB (wash buffer) overnight, by putting the reservoir funnels on the columns and adding 100mL at once.

DAY TWO

12.Let the column go dry briefly. Add 4mL EB and let sit for one minute. Elute the protein with small volumes of EB while checking for protein by Bradford assay (use 10uL of the elute with 200uL of Bradford reagent). Elute until no more protein is present in the eluate.

13.Add EDTA to 1mM final concentration. Wait 5 minutes and add BME to 5mM.

14.Collect an aliquot of the uncut protein for SDS-PAGE analysis (20uL in 20uL sample buffer). One may wish so save a pure sample of uncut protein for crystal trial setups in this case pr degreeeed to step #22.

15.Add His-tagged rTEV protease to the protein samples at the ratio of 60mg enzyme/1g protein, if possible. (Less protease may be used if less is available.) Assay protein concentration by A280.

16.Dialyze the sample in 2-4L of Dialysis Buffer at 4 degree overnight. Do not exceed 40-80 mL of sample to 2-4L of buffer.

DAY THREE


17.Run a SDS-PAGE analysis of the raw cells, lysis pellet, affinity flowthrough, eluted protein before digestion and the digestion reaction (10uL in 10uL sample buffer). Run 5uL of the cells and lysis pellet and 10uL of the rest. If digestion is incomplete, allow the TEV protease to digest further (24 hours or so).

18.Wash and recharge the Ni resins, keeping them separate. The Ni wash prot degreeol is on a separate sheet.

19.Equilibrate the washed, recharged resins with 20mL BB and allow to go dry briefly. When the digestion is complete, apply it to the column with the bottom stopc degreek closed. Allow to sit without flowing for 30-60 minute. Collect the flow-through and add small aliquots of BB until all the cut Protein has passed through as determined by the Bradford assay.

20.Using a sample of the digestion before this second purification as a relative reference to determine if all of the protein passed in the flowthrough in step #19. If protein doesn't flow through use EB to collect the elute as in #12 above.

21.Add EDTA to 1mM, wait 5min and add BME to 5mM.

22.Dialyze the protein in 2-4L of Crystallization Buffer at 4 degree overnight.


DAY FOUR

23.Concentrate the protein up to a volume of 0.2-.05mL (Millipore concentrators). Measure the concentration before and after the concentration by A280. The final concentration should be 5-25mg/mL.

24.Pass the samples through a 0.22um micr degreeentrifuge filter.

25.Distribute the protein in small aliquots (100-200uL), flash freeze in liquid N2, and store at -70 degree.


8 March 2004 / Matthew Zimmerman, Pete Miles / original prot degreeol U. Toronto