核蛋白提取protocol

Cytoplasmic and Nuclear Protein Extraction Materials:
Reagents:
Low Salt Buffer-For 10mL
200µL of 1M HEPES pH 7.9
2.5mL of glycerol
15µL of 1M MgCl2
200µL of 1M KCl
8µL of 250mM EDTA
100µL of 100mM DTT郃DD FRESH!
50µL of 100mM PMSF郃DD FRESH!
6.927mL of dH2O
High Salt Buffer-For 10mL
200µL of 1M HEPES pH 7.9
2.5mL of glycerol
15µL of 1M MgCl2
2.67mL of 3M KCl
8µL of 250mM EDTA
100µL of NP-40
100µL of 100mM DTT郃DD FRESH!
50µL of 100mM PMSF郃DD FRESH!
4.357mL of dH2O
Sucrose Buffer w/o NP-40-For 10mL
3.2mL of 1M Sucrose
300µL of 0.1M CaCl2
20µL of 1M MgAc
4µL of 250mM EDTA
100µL of 100mM DTT郃DD FRESH!
50µL of 100mM PMSF郃DD FRESH!
6.326mL of dH2O
Sucrose Buffer w/ NP-40-For 1mL
1mL of Sucrose Buffer w/o NP-40
5µL of NP-40
Procedure:
PERFORM ALL STEPS ON ICE!
1. Collect cells from 1 confluent T75 (scraping or trypsinizing, doesn抰matter).
2. Wash cells once with ice-cold PBS and repellet.
3. Resuspend cells in 1mL ice-cold PBS and transfer to an eppendorf tube.
4. Pellet cells at 200xg for 5 minutes.
5. Resuspend cells in 200mL Sucrose buffer with NP-40 by gently pipetting with a 1000mL tip, and incubate on ice for 5 minutes to lyse.
6. Pellet nuclei by centrifugation at 1500xg for 5 minutes and transfer the supernatant (cytoplasmic fraction) to a new tube. (NOTE: It抯best to leave the last 50mL at the bottom of the tube out of your cytoplasmic fraction, this reduces the likelihood of contaminating the cytoplasmic fraction with nuclear protein.)
7. Gently resuspend the nuclei in 1mL Sucrose buffer without NP-40.
8. Pellet the nuclei at 1500xg for 5 minutes. Discard supernatant. This and the above step removes leftover cytoplasmic contaminants using a sucrose cushion.
9. Gently resuspend nuclei in 50mL LOW salt buffer (nuclei should be semi-granular, and intact).
10. Add 0.2X volume HIGH salt buffer and gently flick tube.
11. Continue adding 0.2X HIGH salt buffer and gently flicking until 1X volume has been added OR the nuclei begin to shrink and viscosity increases (it generally takes me about 0.4X volume with HeLa cells).
12. Incubate tubes on the rotary platform in the cold room for 20 minutes
13. Centrifuge at 13000xg for 15 minutes.
14. Retain supernatant (nuclear fraction).
QC Controls: β-Tubulin and PARP Western Blots on both nuclear and cytosolic samples
NOTE: This method is low-salt, so it does not disrupt cytoskeletal interactions, which means you WILL pellet most if not all of any cytoskeletal proteins. This includes nuclear cytoskeletal proteins.
HELA CELL NUCLEI PREP
I. Solutions:
a. Chelsky Buffer (200ml)
0.01M Tris-HCl
0.01M NaCl
0.003M MgCl2
0.03M Sucrose (mw. 342.3 g/mole)
pH to 7.0 and bring up to 200ml.
b. NP-40 Buffer
100ml of Chelsky Buffer
500ul of NP-40 detergent
c. Cacl2 Buffer
100ml of Chelsky Buffer
1.0ml 100x stock CaCl2 sol'n (10mM)
d. Buffer C
20mM Tris-HCl pH 7.9
20% glycerol
0.1M KCl
0.2mM EDTA
pH to 7.9
II. Procedure:
a. Cell collection:
Plate Helas on 150mm plates on day 1. (1 x107 cells yields about 1 mg of total cellular protein) Approximately 32-150mm plates.
Trypsinize every plate on day 2-3 (am).
Bring each plate up in 5ml of media, and count the total number of cells\ml. Record the volume of cell suspension.
b. Cell treatment:
Note: Everything (buffers, cells, centrifuge) must be kept on ice (4oC) throughout this entire procedure, to reduce protein degradation.
Note: Save the supernatant from each wash to keep track of protein if lost.
Wash the Hela cells 2x with 25 ml of cold PBS per tube in clinical centrifuge setting #3 in cold room(4oC).
Resuspend cell pellets in 2.0 ml of NP-40 Buffer, spin in HB-40 rotor at 3000rpm (1500xg) for 10mins,remove supernatant and resuspend as above.
Repeat NP-40 wash step above.
Remove supernatant and resuspend pellet into 2.0ml of CaCl2 Buffer. Spin in HB-40 rotor at 3000 rpm (1500xg) for 10mins, remove supernanat and resuspend as above. Repeat CaCl2 wash step.
Resuspend pellet from second CaCl2 wash step into 2.0 ml of Buffer C, depending on how much protein was collected (use 1ml/number of cells) Spin balanced tubes in SS34 rotor at 14,500 rpm(25,000xg) for 30mins.
Collect supernatant (nucleoplasm), resuspend pellet (nuclear envelope) in Buffer C and homogenize to get into solution.
Aliquot into cryovials and freeze at -70oC.
Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots
The following protocol is optimized for about 107cells (near confluent 10 cm plate). NOTE: ALL COMPONENTS (BUFFERS, PROTEASE INHIBITORS, ETC) MUST BE KEPT ON ICE DURING THE ENTIRE PROCEDURE.
1. 1. Wash plates with PBS two times.
2. 2. Prepare 5 ml of Buffer A in a 15 ml conical c’fuge tube, and add the fol lowing:
-5 ul of each protease inhibitor (leupeptin, PMSF, aprotinin, pepstatin, and DTT)
-200 ul of 10% IGEPAL.
Add 0.5 ml of this preparation directly to each plate, and wait 10 min. at room temp.
3. 3. Scrape cells with NEW sterile scraper, then pipet up and down with P1000 several
times to disrupt cell clumps. NOTE: you will have nearly 1 ml of lysate at this point.
4. 4. Transfer this lysate to a 1.5 microcentrifuge tube, place on ice.
5. 5. Centrifuge at 4 C at top speed (15000 x g) for 3 min.
6. 6. Place tubes on ice.
7.7. Save supernatant (cytosolic fraction) for luciferase/ß-gal assay, if desired. Otherwise,
discard the supernatant.
8.8. Prepare 1 ml of Buffer B in a 1.5 ml eppendorf, and add 1 ul of each protease inhibitor
as above, but this time DO NOT add IGEPAL. Add 150 ml of this buffer to each tube, and resuspend pellet by pipeting up and down with a P200. Place on ice.
9.9. Shake vigorously at 4 C for 2h.
10.10. Centrifuge at 4 C, top speed for 5 min. Measure Bradford (protein) concentration using
5 ul of each sample, then aliquot 15 ul aliquots into prechilled 0.5 ml prelabeled
microcentrifuge tubes, freeze in liquid nitrogen, and store in –80 C freezer.
Buffer A:
10 mM HEPES, pH 7.9
10 mM KCl
0.10.1 mM EDTA
Add just before use: 1mM DTT, 0.5 mM PMSF, 5 ul of 10 ug/ul of aprotinin, leupeptin, and pepstatin A to 5 ml of buffer.
Buffer B:
20 mM HEPES, pH 7.9
0.4 M NaCl
1 mM EDTA
10% Glycerol
Add just before use: DTT, PMSF, aprotinin, leupeptin, pepstatin A as above.
Nuclear and Cytoplasmic Extracts
Materials:
• Endonuclease (Sigma E8263)
• 10% Triton X or IGEPAL 630
• Phosphatase inhibitor Solution
• Phosphate buffered Saline (PBS), pH 7.4
• Solution A (see recipe at the end of prot ocol)
• Solution B (see recipe at the end of protocol)
• Solution C (see recipe at the end of protocol)
• DeStreak Rehydration Solution (Amersham-Biosciences)
Method:
1. Wash cell plate with ice-cold PBS. Add 1ml ice-cold PBS with Phosphatase inhibitor solution per 10cm plate and scrape cells. Transfer to an ice-cold Eppendorf tube.
2. Pellet cells at 1000 X g for 2 minutes. Remove supernatant.
3. Add Solution A without resuspending the pellet at a 1:1 (v/v) ratio to that of the cellular pellet. Centrifuge as in step 2. Remove supernatant.
4. Add Solution A at a 1.5:1 volume of Solution A per volume of cell pellet. Briefly vortex
or resuspend pellet, incubate on ice for 10 minutes. Add 1/20th volume [cell pellet + Solution A] of 10% Triton X (or IGEPAL 630) to a final concentration of 0.5% and mix. Centrifuge 6000 rpm @ 4 ºC for 30 seconds.
5. Collect supernatant (cytoplasmic extract). Determine concentration, aliquot into 200 μg samples and store immediately at –80 ºC.
6. Add Solution B in a 1:1 volume relative to nuclear pellet and gently resuspend. Centrifuge 12,000 rpm @ 4 ºC for 10 minutes. Discard supernatant.
7. Add DeStreak Rehydration solution in a 2:1 ratio to the pellet. Vortex for 5
minutes at room temperature.
8. Add endonuclease (Sigma, E8263) to a final concentration of 300 units/ml and incubate at room temperature for 30 minutes.
9. Sonicate the sample for 10 seconds. Centrifuge 13k rpm @ 4 ºC for 30 minutes. Collect supernatant (nuclear extract). Determine protein concentration.
10. Aliquot into 200 μg samples and store immediately at –80 ºC.
Reagents and Solutions:
*DTT and PMSF should be added immediately prior to use
1. Add M.Q. H2O to a final volume of 20 ml.
2. First add DTT then PMSF and quickly vortex vigorously
3. Keep the solution on ice and store at 4°C
Note:
*DTT and PMSF should be added immediately prior to use
1. Add 6.8 grams sucrose (final concentration 1.0 M)
2. Add M.Q. H2O to a final volume of 20 ml.
3. First add DTT then PMSF and quickly vortex vigorously
4. Keep the solution on ice and store at 4°C
C. Solution C (High salt buffer)
*DTT and PMSF should be added immediately prior to use
1. Add M.Q. H2O to a final volume of 2 0ml
2. Add DTT first then PMSF and quickly vortex vigorously
3. Keep the solution on ice and store buffer at 4°C
D. Phosphatase Inhibitor Salt Solution。