Aug 22, 2018
Version 2
His10-MBP-Cas9 purification V.2
- Audrey Lapinaite1,
- Sam Sternberg1,
- Brett Staahl1
- 1University of California, Berkeley
- The Center for Genome Editing and Recording
Protocol Citation: Audrey Lapinaite, Sam Sternberg, Brett Staahl 2018. His10-MBP-Cas9 purification. protocols.io https://dx.doi.org/10.17504/protocols.io.ssseeee
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2018
Last Modified: August 22, 2018
Protocol Integer ID: 14898
Abstract
Attachments
Guidelines
This protocol takes two days to execute.
Materials
MATERIALS
Please see before starting in Guidelines section for materials.
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warnings and hazard information.
Before start
DAY 1 materials
| Lysis Buffer | 200 ml | |
| 50 mM Tris-Cl, pH 7.5 | 10 ml 4M | |
| 500 mM NaCl | 25 ml 4M | |
| 1 mM TCEP | 400 µl 0.5M | |
| 5% glycerol | 10 ml 100% | |
| 0.5mM PMSF | 250 µl 400 mM | |
| protease inhibitors (Roche) | 4 tablets |
| Wash Buffer | 500 ml | |
| 50 mM Tris-Cl, pH 7.5 | 25 ml 1M | |
| 500 mM NaCl | 62.5 ml 4M | |
| 1 mM TCEP | 1 ml | |
| 10 mM imidazole | 1.67 ml 3M | |
| 5% glycerol | 25 ml 100% |
| Elution Buffer | 200 ml | |
| 50 mM Tris-Cl, pH 7.5 | 10 ml 1M | |
| 500 mM NaCl | 25 ml 4M | |
| 1 mM TCEP | 0.4 ml 0.5M | |
| 300 mM imidazole | 20 ml 3M | |
| 5% glycerol | 10 ml 100% |
DAY 2 materials
| Cas9 IEX Buffer A | 1500 ml | |
| 20 mM Tris-Cl, pH 7.5 | 30 ml 1M | |
| 125 mM KCl | 93.75 ml 2M | |
| 1 mM TCEP | 3 ml 0.5M | |
| 5% glycerol | 75ml 100% |
| Cas9 IEX Buffer B | 500 ml | |
| 20 mM Tris-Cl, pH 7.5 | 20 ml 1M | |
| 1 M KCl | 250 ml 2M | |
| 1 mM TCEP | 1 ml 0.5M | |
| 5% glycerol | 25 ml 100% |
| Cas9 Gel Filtration Buffer | 1000 ml | |
| 20 mM Tris-Cl, pH 7.5 | 20 ml 1M | |
| 200 mM KCl | 100 ml 2M | |
| 1 mM TCEP | 2 ml 0.5M | |
| 5% glycerol | 50 ml 100% |
DAY 1
DAY 1
Harvest cells from large-scale overnight cultures by spinning down in 1 L centrifuge bottles: 20 minutes at 4000 rpm, 4 °C.
Note
One of the two centrifuges can only go up to max speed of 3400 rpm.
4 °C Spinning down
00:20:00 Spinning down
Pour off supernatant and either freeze cell pellet (in centrifuge bottle) at -80 °C until purification, or proceed to step #3.
-80 °C Freezing cell pellet
Note
Better to not keep pellets in centrifuge bottles too long, since this will deplete the lab supply.
Resuspend cell pellets in lysis buffer. Use 10-15 ml per 1 L of culture. Multiply accordingly based on the number of flasks you grew. Either use resuspended cells and proceed directly to step #4, or transfer to 50 ml Falcon tubes, flash freeze in liquid nitrogen, and store at -80 °C until purification.
Save 20 μL for SDS-PAGE analysis: “cell pellet”
-80 °C Storage
Lyse cells by sonication. Do not do this in glass containers (they can shatter).
- Sonicate at power setting 6.0: 10 seconds on, 20 seconds off, total run time of 5 minutes. Cells must be on ice during this step, as the temperature will increase during sonication and the cells must stay cold.
00:05:00 total
00:00:10 on
00:00:20 off
Note
Either sonicate directly in 50 ml Falcon tubes, or Oakridge tubes, or for large volumes, in plastic beakers. For small cultures I use the sharp tipped needle on the sonicator, but usually (for resuspended pellets deriving from >2 L of culture) I use the blunt-ended tip on the sonicator. Make sure it is screwed on tightly. Before setting up sonication, wash the tip in H2O and EtOH, and dry before use with Kimwipe.
Clarify cell lysate by centrifugation at 15,000 rpm for 30 minutes, 4 °C.
During the 30 minute spin, equilibrate Ni-NTA resin in wash buffer.
(1) To equilibrate, transfer resin into 50 ml Falcon tube, and add mQ H2O to about the 50ml mark. Invert a few times to fully resuspend the resin. Centrifuge at 2400 rpm (no higher!) for 3 minutes, and gently pour off supernatant into the sink immediately after spin is over. These pellets are quite fragile, and will begin to dissipate in the supernatant rather quickly. Avoid accidentally pouring off your Ni-NTA resin.
(2) Repeat (1) by adding more mQ H2O, spinning, and pouring off supernatant.
(3) Repeat (2) 2-3 times, but using wash buffer instead.
Note
It is critical that tubes are precisely balanced for these rotors spinning at these high speeds. Use the digital balance and balance to within +/- 0.01 g.
4 °C Centrifugation
00:03:00 Centrifugation II
00:30:00 Centrifugation I
Note
To equilibrate Ni-NTA resin in wash buffer, I generally use a column volume of ~5 ml resin for 2-4 L of culture, and then scale accordingly if growing less or more. (Note that the Ni-NTA stock bottles are a 50% (v/v) suspension in EtOH, and so for 5 ml column volume you need to use 10 ml of this suspension.
After the spin in step #5 concludes, save 20 μl for SDS-PAGE analysis: “cell lysate”
Now, transfer entire supernatant (by decanting or with a serological pipette... I prefer a pipette) to the Falcon tube containing your Ni-NTA resin equilibrated in wash buffer.
Note
If volume of supernatant is larger than will fit into one Falcon tube, you can split sample into 2 (or more), being sure that the Ni-NTA resin and your lysate is evenly mixed between each. Be careful to avoid disturbing the cell debris pellet when removing the supernatant.
Rock the Ni-NTA resin with cell lysate at 4°C, 30-60 minutes, in the Falcon tube.
4 °C Rocking Ni-NTA resin
00:30:00 Rocking Ni-NTA resin
Pour Ni-NTA resin with lysate into disposable plastic (or glass) column. If using a glass column, be sure to thoroughly wash with copious amounts of EtOH and water before use.
Save 20 μl of the flow-through for SDS-PAGE analysis: “flow-through, FT.” Aside from this, the flow-through will not be saved and so can be collected in a waste container.
Note
During steps #9-12, also monitor the amount of protein in each fraction via the Bradford assay.
a) Bradford reagent undergoes a color change in the presence of protein. It can be used to do quantitative protein concentration measurements, or in this case, give us a quick colorimetric read-out for: if our washes succeeded than there should be no protein coming off the column during wash #5, and so no color change. Similarly, Bradford assay will tell us which of our elution fractions contain a substantial amount of our protein of interest (or any protein at all!)
b) Note that one could also use the A280nm reading to determine protein concentration, but it’s more work at this stage then necessary.
Wash the resin with 5 column volumes.
Save 20 μl of the flow-through for SDS-PAGE analysis: “wash #1, W1.” Aside from this, the washes will not be saved and can be collected in a waste container.
Repeat step #10 4 more times, for a total of 5 washes.
Save 20 μl of the flow-through for SDS-PAGE analysis: “W2, W3, etc.” Aside from this, the washes will not be saved and can be collected in a waste container.
Elute by adding 1 column volume of elution buffer, 5 times. Be sure to collect these fractions which contain our protein of interest! Collect each elution fraction in a separate 15 ml Falcon tube.
Save 20 μl of each elution fraction for SDS-PAGE analysis: “elution #1, E1, E2, etc.”
Based on Bradford, pool the elution fractions that contain protein. Measure the absorbance at 280 nm and record this, along with the volume, to give us the overall yield. Also, print out the absorbance spectrum for your notebook, since this has information on the purity of protein (or, if it is co-purifying with any nucleic acid).
If cleaving off the His10-MBP fusion, add TEV protease to elution fractions.
Note
The TEV protease purified by Kaihong is typically 0.5 mg per tube (250 μl); use 1 tube for every 10–20 mg of purified protein.
After adding TEV protease to the eluate, dialyze overnight in Wash Buffer using a Slidealyzer cassette, MWCO = 10k or 30k. Make sure the buffer is pre-chilled to 4 °C.
DAY 2
DAY 2
Run TEV-cleaved Cas9 back over the Ni-NTA resin, being sure to equilibrate the resin in Wash Buffer first (>10 CV). Collect the flow-through. Add 1 column volume of wash buffer and collect with the flow-through.
Place TEV-cleaved Cas9 back into a Slidealyzer cassette, and dialyze into IEX Buffer A for 2– 4 hours.
Note
It is critical that this dialysis not proceed longer, as Cas9 is not very soluble at low salt and can will precipitate with prolonged incubation. (The disadvantage of raising the salt concentration is that Cas9 will no longer bind the SP column efficiently.)
02:00:00 Dialysis
Purify Cas9 by ion exchange, using a HiTrap HP SP column. Protocol: 0–100% Buffer B over the course of 20 column volumes. Cas9 should elute at ~26 %B. Concentrate to <2 ml.
Purify Cas9 by gel filtration, using a Superdex 200 16/60 column. Concentrate and snap freeze.