PULLDOWN AND SOLUBILITY TEST

Lysis buffer (for all but the Nickel Column)

50 mM Tris HCl pH 7.5
50 mM NaCl
5 mM BME

Lysis Buffer for Nickel Column

50 mM Tris HCl pH 7.5
0.5 M NaCl
(no BME!)

Nickel Column Buffer A

20 mM Tris HCl pH 8.0
0.5 M NaCl
50 mM immidazole pH 8.0

Nickel Column Buffer B

20 mM Tris HCl pH 8.0
0.5 M NaCl
0.5 M immidazole pH 8.0

CELL GROWTH AND LYSIS

1. For your vector of choice (or all of them), transform BL21 cells with your plasmid. Use 1-2 μL of DNA to 200 μL BL21
cells. Plate these cells on LB/Amp plates. Grow overnight.
2. Start 2-50 mL cultures (50 mL LB + 50 L ampicillin) with two different BL21 colonies. Grow at 37 ˚C until one of the
cultures has an OD600 of 0.4. Discard the other samples.
3. Take a sample with a volume = 0.4/ OD600 (mLs) and place in an eppendorf. Spin for 10 minutes at 13,200 rpm. Aspirate
off the supernatant and resuspend the pellet in 100 L of 1x SDS buffer. This will be a sample of the uninduced protein.
When you are ready to run your gel, boil the sample for 8 minutes so that the cells are fully lysed.
4. Move the 50 mL culture that is at an OD600 of 0.4 to the room temperature shaker. Induce with 0.5M IPTG and grow for 4
hours.
5. Take a sample with a volume = 0.4/ OD600 mLs and place in an eppendorf. Spin for 10 minutes at 13200 rpm. Discard the
supernatant and resuspend the pellet in 100 L of 1xSDS buffer. This will be a sample of the induced protein. When you
are ready to run your gel, boil the sample for 8 minutes so that the cells are fully lysed.
6. Pellet the cells from the 50 mL culture in two small centrifuge tubes (JA-20) using either the Sorvall or the Beckmann.
Spin at 7000 rpm for 10 minutes. If necessary freeze the pellets in the –80 ˚C freezer overnight.
7. Dissolve the pellet from 25 mL of the culture in 2 mL of lysis buffer. (NOTE: verify that the protein pI is not too close to
pH 7.5 – 8.0.) NOTE: If doing a Nickel pulldown, lyse the cells in the buffer without BME.
8. Lyse the cells using 30 μL of 10 mg/mL lysozyme (make fresh from powder stored in –20 freezer). Incubate on ice for 20
minutes with the lysozyme.
9. Sonicate the cells three times for 15 seconds at 50% output and 50% power. Keep the cells on ice between sonification
rounds so that the cells do not get hot.
10. Transfer the cells to a 2 mL eppendorf tube. Spin the cells down at 13,200 rpm for 30 minutes in cold room.
11. Save the supernatant in a new 1.5 mL eppendorf. Take 100 μL of the supernatant and add 100 μL of 2X SDS buffer. Boil
the sample for 8 minutes. Spin for 5 minutes. This will be your supernatant sample. Reserve 0.5 mL of supernatant for
the pulldown experiment (see below). Keep the remaining supernatant at 4 C until procedure is complete.
12. Resuspend the pellet in a volume of lysis buffer equal to that of the supernatant. Take 100 μL of the redissolved pellet and
add 100 μL of 2X SDS buffer. Boil the sample for 8 minutes so that the cells are fully lysed. Spin for 5 minutes. This
will be your pellet sample.

GST PULLDOWN (pGEX vectors)

1. Add 100 L of GST bead slurry to a 1.5 mL eppendorf.
2. Wash beads 3x with 1 mL of ddH2O. To wash, add the water to the eppendorf. Mix by inverting the tubes several times.
Spin the tube down with a table-top centrifuge for ~15 seconds. Pour out the water. Be careful not to pour out the beads.
3. Next wash the beads 3x with lysis buffer.
4. Add 0.5 mL of supernatant (prepared as above).
5. Incubate on ice for 30 minutes. Every times minutes, flick the tube to mix.
6. Take a 50 L sample of the supernatant after the 30 minute incubation (this will test how much the protein did not absorb
on the beads). Add 50 L of 2x SDS buffer.
7. Discard the remaining supernatant.
8. Wash the beads 5x with 1 mL of lysis buffer.
9. Add 100 L of 2x SDS buffer to the beads. Boil for 8 minutes and spin down the beads. The sample is ready to run on a
gel.

MALTOSE BIND PROTEIN PULLDOWN (pMAL vectors)

1. Add 100 L of maltose binding bead slurry to a 1.5 mL eppendorf.
2. Wash beads 3x with 1 mL of ddH2O. To wash, add the water to the eppendorf. Mix by inverting the tubes several times.
Spin the tube down with a table-top centrifuge for ~15 seconds. Pour out the water. Be careful not to pour out the beads.
3. Next wash the beads 3x with lysis buffer.
4. Add 0.5 mL of supernatant (prepared as above).
5. Incubate on ice for 30 minutes. Every times minutes, flick the tube to mix.
6. Take a 50 L sample of the supernatant after the 30 minute incubation (this will test how much the protein did not absorb
on the beads). Add 50 L of 2x SDS buffer.
7. Discard the remaining supernatant.
8. Wash the beads 5x with 1 mL of lysis buffer.
9. Add 100 L of 2x SDS buffer to the beads. Boil for 8 minutes and spin down the beads. The sample is ready to run on a
gel.

MINI-NICKEL COLUMN
To charge column (if column is not blue in color):
1. Get the small nickel column.
2. Wash it with 5 mL of ddH2O using a syringe.
3. Charge it with 0.5 mL of nickel solution.
4. Wash with another 5 mL of ddH2O.

To run column:
1. Wash with 5 mL of Nickel Column Buffer A.
2. Load 2-3 mL of the protein supernatant.
3. Wash with 10 mL of Nickel Column Buffer A.
4. Elute the protein with 5 mL of Nickel Column Buffer B. Collect the flow through in 1 mL aliquots.
5. Wash with another 10 mL of Nickel Column Buffer B.
6. Wash with 10 mL of Nickel Column Buffer A.
7. Run a gel on all the aliquots of eluted protein.

GELS
1. Run a SDS page gel using uninduced, induced, supernatant, pellet samples, supernatant after bead binding and beads
(pulldown). The % of polyacrylamide depends on the size of your protein. Run the gel at 70 V with a broad range marker.
Use 6 μL of marker and 15 μL of the samples.

UNIND IND SUPERNATANT PELLET SUPER POST BINDING PULLDOWN

For the nickel pulldown:

UNIND IND SUPERNATANT PELLET ELUTION ALIQUOTS (5)

2. Develop the gel as follows:

 Remove gel from mold and place in fixer for 5 minutes (place on room temperature shaker)
 Dump fixer and add stain/destain plus commassie blue stain
 Microwave for 2 minutes
 Dump stain and add destain
 Place on shaker for several hours or until fully destained
3. Verify that you have the correct size protein and that the induction worked.
4. Check the solubility of the protein by comparing the amount of protein in the supernatant verse the amount in the pellet.
5. Based on the protein solubility in the different vectors, determine which one is most appropriate for proceeding with
purification.