Alpha-B Core Domain Protocol
Updated: 9/2/11
Growth
1. Induce cells until OD of 0.45-0.6
2. Grow at 22C overnight
Lysis
1. Spin down cells at 5k rpm in SS34 tubes for 30 minutes
2. Resuspend cells in 20 mM Tris 200 mM NaCl pH 8.0 (15 mL/1L growth)
3. Add 1 aliquot HIS-protease inhibitor cocktail (per 20 mL lysate) and 60 uL
PMSF (43 mg/mL EtOH)
4. Add MgCl2 to 10 mM and small amounts of DNase and RNase.
5. Run cells though the French press cells twice; add PMSF after each press.
6. After pressing the cells, aliquot cells into 20 mL fractions in 50 mL conical vials.
7. Add 6M Urea, 200 mM NaCl until sample volume reaches 50 mL.
8. Spin Urea lysate at 17k rpm with SS34 rotor for 20 minutes.
Ni-Column [edit for large volumes]
1. Equilibrate Ni2+ column with ddH2O and 6M Urea, 200 mM NaCl.
2. Add the cell lysate supernatant to the ni2+ column and let the volume drop to
½ the volume of the plastic column.
3. At this point, refill the column with ni2+ start buffer; repeat this 2 times.
4. Let the supernatant volume drop to the resin bed and add 5 mL nickel start
buffer.
a. Collect the above refold steps as the FT for SDS-PAGE.
5. Perform a 15 mL 50 mM imidazole wash
a. Collect a sample for SDS-PAGE.
6. Perform a 10-15 mL 250 mM imidazole elution
a. Collect a sample for SDS-PAGE.
7. Perform a 10-15 mL 500 mM imidazole elution (protein should also be in this
fraction in large amounts).
a. Collect a sample for SDS-PAGE.
8. Run a 15% gel on the fractions.
a. Keep 250 mM and 500 mM Imidazole elutions separate if desired.
TEV Digest
1. Pool clean fractions containing alpha-B (sometimes the 50 mM imidazole
wash is pretty clean).
a. Check this box if the 50 mM Imidazole fraction was used.
2. Concentrate the volume to ~10 mL (R120G does not like this step, so it may
be best to avoid it entirely).
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Alpha-B Core Domain Protocol
Updated: 9/2/11
3. To a 10-15 mL core domain volume, add 3 aliquots 1 mg/mL TEV protease
(each aliquot is 1 mL).
4. Add PMSF to 50-100 uL per 15 mL core domain volume.
5. Dialyze the sample overnight at 4C in 20 mM Tris, 100 mM NaCl pH 7.6-8.0
dialysis buffer.
a. 12.6g Tris, 23.376g NaCl, pH 7.6 (makes 4 L)
Ni2+ Column – Day 2
1. Run a gel on the digest in order to get an idea of the digest efficiency. 80% or
better is what you want to see. Add more TEV if you don’t see this and let it
digest for a few more hours.
a. NOTE: You may observe a precipitate in your dialysis tubing caused by
addition of PMSF. Do not fret! While it is unclear what this precipitate is,
it is likely either PMSF or a small amount of your overall protein sample.
Either way, it will not reduce your final yield by any significant amount.
i. Centrifuge away any precipitate on the table-top centrifuge at
3700 rpm for 4 minutes.
2. Pour the digest over an equilibrated Ni2+ column and collect the FT.
a. Collect a sample for SDS-PAGE.
i. PROTEIN SHOULD BE IN THIS FRACTION.
3. Do 2x 50 mM imidazole washes at 10 mL per wash and collect as separate
fractions.
a. Collect samples of each one for SDS-PAGE.
i. PROTEIN SHOULD BE IN THIS FRACTION.
4. Perform a 250 mM imidazole elution at about 10-15 mL.
a. Collect a sample for SDS-PAGE
5. Perform a 500 mM imidazole elution at about 10-15 mL.
a. Collect a sample for SDS-PAGE.
6. Add PMSF to all fractions and run a gel to determine location of protein.
G-25/MonoQ
1. Take the FT and wash fractions that contain clean protein and run them over
the G-25 column on “Captain.”
a. G-25/Captain need to be in 20 mM Tris pH 8.0 (2 L)
2. Pool fractions contain protein (determined via the readout on the computer
monitor).
3. Run protein over the MonoQ.
a. Methods for the wt and r120g are located in scott’s folder.
b. Line A should be in 20 mM Tris pH 8.0 (1 L)
c. Line B should be in 20 mM Tris, 1 M NaCl pH 8.0 (1 L)
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Alpha-B Core Domain Protocol
Updated: 9/2/11
4. After completion of the column run, add 1 uL of PMSF to ALL fractions
containing protein and store at 4C overnight.
5. Begin equilibrating an SDX75 column overnight for tomorrow.
a. Line A should be in 50 mM Pi, 250 mM NaCl, pH 7.0
6. Run a 15% on ALL fractions that you are considering pooling the next
morning.
7. Examine the gel and pool the cleanest fractions. Make sure to omit any
fractions that contain a lot of contaminates.
SDX75
1. On any of the medium SDX75 columns, size your protein and add 1 uL PMSF
to fractions.
2. Run a gel on all fractions you are considering pooling in order to assess purity.
3. Pool clean fractions containing your alpha-B core domain protein.
a. It is OK to omit pooling the “tailing” fractions (based on your elution
profile), or to pool them separately as a “not-as-good” pool. While they
may look clean on the gel, it is unclear why they “tail” off the column.
b. Be sure to check for degradation on this gel; if alpha-B degraded, it
would be wise to perform another monoQ run. This SHOULD NOT happen
if you were efficient during the course of this protein prep.
4. If the protein is not clean after looking at the gel, run the sample over the G25 and MonoQ again (this should clean it up).
5. Determine amount of protein obtained in milligrams via UV vis.
Dialysis/Lyophilization
1. Dialyze protein overnight into 4 L of either 1 mM Pi or H2O (determine which
one by asking the person you are preparing the protein for).
2. Dialyze protein again in the same buffer the following morning for ~3 hours.
3. Lyophilize protein (ask someone to show you how to do this if you are
unsure).
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