Analyzing native, intact complexes by conventional chromatography methods is not always successful or practical. Using an antibody directed against a subunit of a complex or inserting an epitope tag into a complex subunit achieves a much more sensitive method for purification of intact complexes. For even greater precision, the use of two affinity tags can be used to purify complexes to homogeneity.
We will use FLAG-affinity purification (general: protein tag) to isolate a RNA Polymerase II C-terminal domain kinase complex from 293FRT/TREx cells. 293FRT/TREx cells have been stably transfected with a plasmid carrying FLAG::CDK9 under the control of a tetracycline-regulated CMV promoter. We will prepare nuclear extract from these cells and incubate our lysate with FLAG antibody-agarose beads. The FLAG epitope in the FLAG::CDK9 fusion binds to the FLAG antibody-conjugated agaraose beads. After several washes to remove unbounded proteins, we will elute the complexes associated with CDK9 from the beads using a large molar excess of FLAG peptide. We will then analyze the purified complex by SDS/PAGE followed by silver staining. In a later procedure, we will also use our isolated complexes in a kinase assay.
Outline:
A. Purification of FLAG-tagged Protein Complexes for Mammalian Cells
- Nuclear-Extract Preparation
- FLAG Purification
- Appendix
B. Silver Stain
- Silver Stain
- Anticipated Results
- Appendix: Solutions
A. Purification of FLAG-tagged Protein Complexes from Mammalian Cells
Notes:
- Keep all buffers and equipment cool (4C).
- Wear gloves.
- Do all of the steps for both the control (untransfected) 293FRT/TREx cells and the hCdk9 cells in parallel.
A1. Nuclear-Extract Preparation [1]
** Steps #1 and #2 have already been performed by Michael Ugarenko at Northwestern University
i. Harvest control HEK293 cells and HEK293 cells expressing Flag-tagged CDK9. Generally, one confluent 850 cm^3 roller bottle will yield ~3×10^8 cells which corresponds to ~1 mL packed cell pellet.
ii. Wash cells twice with PBS (check the pellet volume at this step), then remove the PBS and snap freeze in liquid nitrogen. Store at -80C until you’re ready to perform the nuclear extract/IP.
iii. Measure cell pellet volumes. You will need approximately 7 mL of Buffer A + 0.5x Protease Inhibitor + 0.5 mM DTT per mL of cell pellet. For GENE-X 2017, cell pellets are ~2 mL, so: 2 mL x 2 pellet x 7 mL Buffer A = 28 mL Buffer A (+ 14 uL 1 M DTT, + 140 uL Sigma Protease inhibitor #8340). Note: Make 2-4 mL of extra Buffer A, you will need it again in the next section: step ii.
iv. Thaw cells on ice. Keep samples on ice throughout the protocol.
v. Resuspend each cell pellet in 5x pellet cell volume (PCV) of Buffer A (+PIC/+DTT). 5x 2 mL = 10 mL Buffer A.
vi. Incubate one ice for 15 min.
vii. Centrifuge: 1,500xg, 5 minutes, 4C.
iix. Aspirate supernatant.
ix. Resuspend each cell pellet in 2x PCV of Buffer A (+PIC/+DTT). 2x 2 mL = 4 mL Buffer A
x. Break cell membranes by homogenization with a glass homogenizer. Use the TIGHT pestle, and 5-10 gentle stokes to disrupt the cells. The extent of cell lysis can be monitored by trypan blue staining. (If you don’t have access to glass homogenizer in your own lab, 0.1 % Triton X-100 also works)
xi. Centrifuge 3,200xg, 20 minutes, 4C.
xii. At this point, the supernatant contains cytosolic proteins and the pellet consists of nuclei. In this instance, discard the supernatant and keep the pellet. Be careful when aspirating, because the nuclear pellet may be loose. If you’re interested in cytoplasmic proteins you can keep a sample of the supernatant.
xiii. Resuspend the nuclei in 0.75x PCV of Buffer C (+PIC/+DTT). 2 mL x 0.75 = 1.5 mL Buffer C
xiv. After resuspending the nuclei, measure the total volume of the suspension (V). Then calculate the volume of 5 M NaCl (N) required to achieve a 420 mM concentration of NaCl.
V = Volume of nuclei suspension after resuspension in Buffer C (usually ~ 3 mL)
N = Volume of 5 M NaCl (N) required to achieve a 420 mM concentration of NaCl
(5 x N)/(V + N) = 0.420
5 x N = (0.420 x V) + (0.420 x N)
4.58 x N = 0.420 x V
N = 0.420/4.58 x V
N = 0.0917 x V
xv. Pipette the required volume of 5 M NaCl into a 1.5 mL tube.
xvi. Add 5 M NaCl to the nuclei suspension in 20 uL increments. Mix the sample gently after each addition. Note that as the NaCl concentration gets higher the sample will become more and more viscous. Pop quiz: Why does the sample get viscous as the NaCl approaches 420 mM?
xvii. Once all the 5 M NaCl has been added, incubate sample on ice for 30 minutes with occasional inversion (or put on rotator in a cold room).
xviii. Pellet the insoluble chromatin pellet by ultra-centrifugation (Make sure to balance tubes!!): Tube: Beckman Coulter 355630, 70.1-Ti Rotor, 40,000 rpm, 60-90 minutes
A2. FLAG Purification
i. Collect the soluble nuclear extract and measure the volume (usually ~ 2 mL).
ii. Dilute NaCl concentration to 300 mM using Buffer A. Remember, the starting concentration from step xvi. is 420 mM NaCl.
V = Volume of Nuclear extract (usually ~ 2 mL)
A = Volume of Buffer A required to dilute NaCl to 300 mM
(0.42 x V) / (V + A) = 0.3
(0.42 x V) = (0.3 x V) + (0.3 x A)
(0.42 x V) – (0.3 x V) = (0.3 x A)
0.12 x V = 0.3 x A
0.4 x V = A
iii. Add 1 uL of Benzonse nuclease to digest any DNA and RNA in the sample.
iv. For each IP transfer 200 uL of anti-Flag-M2 agarose beads into a 15 mL conical tube and wash twice with 1 mL Buffer A (spin at 500xg). Note: pipette beads with a cut or wide-bore tip pipette.
v. Add nuclear extract to the washed beads and incubate overnight at 4C on a rotator. In general Flag-IPs require at least 2-3 hours of binding.
vi. Wash beads 4 times with 5 mL Wash Buffer (Spin 500xg for 5 minutes to pellet beads. Rotate at 4C for 5 min, for each wash step.)
Optional step: Retain first unbound fraction, in case antibody binding doesn’t work.
vii. Prepare 1 mL of Elution Buffer (you will need 400 uL for each IP, 1 mL gives you a little extra)
1 mL Elution Buffer Base
5 uL of Sigma protease inhibitor #P8340
10 uL of 10 mg/mL Flag Peptide (100 ug/mL final concentration)
viii. Elute Flag-tagged protein by incubating beads in 100 uL Elution Buffer for 5-10 min on ice.
xi. Spin down beads (500xg, 5 min) and carefully collect the supernatant and transfer to a new tube (This is Elution #1).
(Alternatively, spin sample through a Bio-Rad Micro Bio-Spin Chromatography Columns #7326204).
x. Repeat steps xi. and x. three more times (a total of 4x 100 uL elution).
xi.At this point you should have 8 tubes: Control (Elution 1-4) and Flag-Cdk9 (Elution 1-4).
xii. To prepare the samples for SDS-PAGE and silver staining, remove a 30 uL aliquot of each elution add 10 uL of 4x SDS Buffer and heat to 95C for 5 min. These can either be used immediately or stored at -20C. DO NOT add SDS to the remaining sample, you want to maintain the Cdk9 in an active conformation.
xiii. Snap-freeze the remaining elutions in liquid nitrogen and store at -80C until ready for the 32P CTD kinase assay (Section B)
A3. Appendix
Buffer A – for 1 L
10 mM HEPES (pH 7.9) – 10 mL 1 M
1.5 mM MgCl2 – 1.5 mL 1 M
10 mM KCl – 4 mL 2.5 M
Just prior to use for the experiment:
Add 1:200 Protease inhibitor #8340 (0.5x final) and 1:2000 1 M DTT (0.5 mM final)
Buffer C – for 1 L
20 mM HEPES (pH 7.9) – 20 mL 1 M
25 % Glycerol – 250 mL 100%
1.5 mM MgCl2 – 1.5 mL 1 M
0.2 mM EDTA – 400 uL 0.5 M
Just prior to use for the experiment:
Add 1:200 Protease inhibitor #8340 (0.5x final) and 1:2000 1 M DTT (0.5 mM final)
Washing Buffer – for 1 L
10 mM HEPES (pH 7.9) – 10 mL 1 M
1.5 mM MgCl2 – 1.5 mL 1 M
300 mM NaCl – 60 mL 5 M
10 mM KCl – 4 mL 2.5 M
0.2% Triton X-100 – 2 mL
Elution Base Buffer – for 100 mL
10 mM HEPES (pH 7.9) – 1 mL 1 M
10% Glycerol – 10 mL 100%
1.5 mM MgCl2 – 150 uL 1 M
100 mM NaCl – 2 mL 5 M
0.05% Triton X-100 – 50 uL
Just prior to use for the experiment:
Add 1:200 Protease inhibitor #8340 (0.5x final)
Elution Buffer
Add 1:100 10 mg/mL of Flag peptide (Sigma F3290)
Notes:
– For analyzing the IP performance, you can use 5 uL of each elution, the S-100, the nuclear extract, and the unbound fraction for immunoblotting with anti-Flag mAb.
– You can make sure you have gotten rid of any carry-over Flag-agarose matrix by passing elutions through a Micro Bio-spin column (1000 rpm x 30 s).
– For Flag Western analysis load 20 uL for each lane. Suggested sample line-up is Marker, S100, Input (Nuclear Extract), Unbound, F-CDK9 IP eluates 1-4. Primary antibody is anti-Flag mAb at 1:2000. Secondary is anti-mouse IgG at 1:10000.
B. Silver Stain
Notes:
- Wear clean gloves and avoid touching parts of the gel that contain your sample.
- Keep all the working solutions at room temperature.
- Do not rinse/wash the gel before fixation.
- Follow the bold-italicized timings exactly.
- Prepare the italicized solutions freshly.
- Decanting of water or solutions at the end of each step should be prompt by gentle holding of an unimportant area of the gel against the tray.
- All chemical solutions and first washes go into the Silver gel waste container, not down the drain, most of these solutions are toxic to aquatic lifeforms.
B1. Silver Stain [1]
i. Thaw Control and Flag-CDK9 elution samples containing SDS loading buffer (section A, step xii.)
ii. Prepare a 4-20% SDS-PAGE gel by cutting along the black line and removing the plastic strip. Assemble the gel running tank using a buffer dam, or share the tank with another group. Fill the inner chamber completely with 1x SDS running buffer and fill the outer chamber until the buffer is halfway up the gel. Remove the comb and gently rinse the wells by pipetting if necessary.
iii. Dilute the BioRad Precision Plus molecular weight marker 1:500 with SDS sample buffer. Silver stain is very sensitive, so if you load undiluted ladder the signal will be much too strong. Load 5 uL of diluted molecular weight markers and 20 uL of each elution fraction.
iv. Perform electrophoresis for 90 min at 20 mA per gel (0.04 A).
v. Fix the gel by rocking in fixing solution (50% ethanol, 10% acetic acid) at room temperature overnight (or for > 1 hour). Cover the gel with plastic wrap to reduce evaporation.
vi. Discard fixing solution and incubate in 30% ethanol for 15min to rehydrate the gel.
vii. Wash the gel with ddH2O 3 times for 5 min. Meanwhile, prepare the Sensitizing (0.02 g / 100 mL Sodium Thiosulfate) and Staining (0.2 g / 100 mL Silver Nitrate) solutions.
viii. Sensitize the gel with the Sensitizing solution for 1.5 minutes. MAKE SURE to keep 2 mL of the Sensitizing solution for preparing the Developing solution.
ix. Wash the gel with Milli-Q water 3 times for NO MORE THAN 30 seconds each time. This step should only take 2 minutes in total.
x. Stain the gel by incubating it in the Staining (silver nitrate) solution for 25 minutes (up to 1 hour). Meanwhile, prepare the Developing solution (100 mL ddH2O, 6g anhydrous sodium carbonate, 2 mL Sensitizing solution, 50 uL 37% Formaldehyde) and the Stop solution (10% Acetic Acid).
xii. Decant the Staining solution and rinse 3 times for 30 seconds intervals with prompt filling and decanting of the gel tray with Milli Q water (This step should only take 2 minutes total).
xiii. Develop the gel by gently shaking in fresh Developing solution while monitoring it until the protein bands are easily visible (this can take between 2-10 minutes).
xiv. Stop developing by discarding the Developing solution and incubate in Stop solution for 10 minutes.
xv. Wash the gels with 4 changes of Milli-Q water.
xvi. Acquire an image of the gel.
B2. Anticipated Results
B3. Appendix: Solutions
Fixing Solution – for 100 mL
Ethanol – 50 mL (50% final)
Glacial Acetic Acid – 10 mL (10% final)
ddH2O – 40 mL
Sensitizing Solution – for 100 mL
Sodium Thiosulfate – 20 mg
ddH2O – 100 mL
Staining Solution – for 100 mL
Silver nitrate – 200 mg
ddH2O – 100 mL
Developing Solution – for 100 mL
Anhydrous Sodium Carbonate – 6 g
Sensitizing Solution – 2 mL
37% Formaldehyde – 50 uL
ddH20 – 100 mL
Stop Solution – for 100 mL
Acetic Acid – 10 mL
ddH2O – 90 mL
SDS-PAGE Run Buffer – for 4 L
10 x Laemmli Running Buffer – 400 mL
ddH2O – 36000 mL
GeneX CSHL 