Protocol

Abbreviation

standard purification_ANL

Name

standard purification_ANL

Laboratory name

Argonne National Laboratory

Type

purification

Description

Day 1 – Small Culture (50mL)

Pick up cell culture from cloning lab. They will be in small, 2mL test tubes. Each tube contains a different target protein cloned in pMCSG7 (or other MCSG) vector and expressed in E.coli BL21 (DE3) cells.

1. Prepare M9 “Pink” Media by adding the following in a 1L bottle:

• 1 pouch M9 Salts + NIAAC
• 10 mL Metal Supplement solution
• 1 mL Kan/Amp/B1/B12 solution
• 10 mL of 50% glycerol
• 980 mL MQ water

2. Aliquot 50 mL of M9 “Pink” Media in small, 250mL-plastic bottle
3. Add 100 uL of LB cell culture
4. Shake in incubator overnight, 37 ºC, 150 rpm

Day 2 – Large Culture (1 L)

1. Prepare M9 “Pink Media” as shown above, except add 930 mL of MQ water
2. Add 25 mL of small culture from small plastic bottle to each 1 L bottle. Place an absorbent underpad on the cart prior to the transfer of bacterial cultures (e.g. Fisher # 14-206-63)). See special section at the end of this document for handling spills for this step.
3. Shake in incubator at 37 ºC, 200 rpm until cells grow to OD 1.0 -1.4. You should start measuring OD (595 nm) after about 2.5-3 hours.
4. Add 30 mL of IAAC + Se-Met solution
5. Transfer bottles to cooled 4 ºC incubator and shake for 1 hour
6. Increase incubator temp to 18 ºC; induce the cells by adding 0.5 mL of 1 M IPTG
7. Grow cells at 18 ºC overnight

Day 3 – Harvesting Cells

1. Pour cell culture in 1L centrifuge bottle. Centrifuge for 8 minutes @ 7000 rpm, SS-6000 rotor (use the cart covered with an absorbent pad for liquid handling and see special section at the end of this document for handling spills)
2. Dispose supernatant into 1:10 diluted bleach solution. Pour into sink after 20 minute incubation.
3. Keep cells in 4 ºC from now on
4. add 1 tablet of Protease Inhibitor
5. Re-suspend the cells by adding 25 mL of cold Lysis Buffer and gentle shaking
6. Transfer resuspended cells into 50 mL Falcon tube and bring volume up to 40 mL with lysis buffer
7. Freeze cells in -80 ºC freezer until they are ready to be purified

Day 4 – IMAC 1*
*Note: AKTA must be stripped, charged, and blanked before actual purification run
1. Thaw out cells - place in bucket of warm water for a few minutes. When thawed, transfer to ice (4 ºC )
2. prepare Lysozyme stock solution: 50 mg/mL. Add Lysozyme to cells @ final concentration 1mg/mL
3. Mix gently and keep cells on ice for 30 - 60 min
4. Sonicate cells on ice for 3 minutes. Program: 4 seconds “on” 12 seconds “off”. Wear ear muffs during the procedure. After the sonication process is complete wait 5 minutes before opening the sonicator housing. Wipe the sonicator tip with gauze (wet the gauze with 10% bleach, allow 20 minutes of contact time), rinse the tip with distilled water and wipe. Discard the gauze in an autoclave bag.
5. Transfer to 45 mL centrifuge tubes. Centrifuge for 1 hour @ 17,355 rpm, SS-34 rotor. Place an absorbent pad on the benchtop or cart during liquid transfer.
6. Using 0.4 μm syringe filter, filter supernatant into clean, 50 mL Falcon tube
7. Run Purification Program in AKTA Xpress

Buffers for AKTA Xpress

Lysis Buffer Elution Buffer Desalting Buffer
HEPES pH 8.0 50 mM 50 mM 50 mM
NaCl 500 mM 500 mM 500 mM
Glycerol 5 % 5 % 5 %
*β-mercaptoethanol 10 mM 10 mM 10 mM
*Imidazole 20 mM 250 mM none

*4X Buffer A does not contain these ingredients




To Prepare Buffers – add ingredients, stir, filter and store at 4 ºC

Stock Solution Lysis Buffer
(4L) Elution Buffer
(2L) Desalting Buffer
(4L)
4X Buffer A 1 L 500 mL 1 L
14.3 M β-mercaptoethanol 2.8 mL 1.4 mL 2.8 μL
2.5 M Imidazole 32 mL 200 mL None
MQ Water Bring up to proper volume

Other Stock solutions needed for AKTA Xpress:
50 mM EDTA/0.5 M NaCl
100 mM NiSO4

Please use the laminated sheet and indicate the protein identifier(s) and your name for those targets of unknown function of RG2 microbes. The sheet must be attached to the chromatography refrigerator where the purifcation procedure takes place.

Day 5 – TEV Cleavage
1. Collect and combine fractions, keep everything at 4 ºC
2. Take 50 uL of each pure protein sample. This will be the “uncut” samples – proteins that still contain His tags.
3. Calculate amount of TEV protease needed
4. Add TEV to remainder of protein sample
5. Incubate proteins at 4 ºC refrigerator for 1-3 days
6. Run SDS-PAGE to determine if tag cleavage has been successful. Use “uncut” protein samples as a reference point.
7. If successful, run IMAC 2

Day ? – IMAC 2

1. Add 4 mL of Ni-NTA slurry to a plastic disposable column
2. Allow column to “pack” by letting liquid flow through
3. Wash packed column with 20 mL MQ water
4. Equilibrate with Lysis Buffer
5. Slowly add protein sample using transfer pipette
6. Collect flow-through sample right away
7. Measure volume and concentration of protein