실험기법

Protein Extraction Protocols

Cell Culture

When extracting proteins from cells the first step is cell culture.

Forskolin Treatment

Application

Mouse NIH/3T3 culture cells can be treated with forskolin, using the following method, to be used as a positive control for anti-phospho-CREB, Catalog No. 06-519.

Method

• Wash cells with PBS at room temperature.
• Dilute a stock solution of forskolin in DMSO with serum-free DMEM media to a final concentration of 50 :M. Caution: Forskolin is soluble only in DMSO. Slowly and carefully add into media to avoid precipitation. WEAR GLOVES!
• Treat cells with forskolin solution for 20 minutes in a 37℃ incubator.
• Rinse cells with ice-cold PBS.
• Add 1 mL ice-cold lysis buffer per 107 cells and process to cell lysate as usual.

Cell Lysate Extracts-General Protcols

There are numerous methods of cell stimulation and lysis. For a given protein, Millipore's laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Please note that in addition to whole cell lysates there are also membrane and nuclear specific preparations that can be used to isolate and concentrate different cellular components. Please keep in mind that cellular stimulation for extended periods of time must be done using sterile reagents and sterile technique. Always observe the cells microscopically before proceeding with a stimulation or lysis protocol to assure that the cells are viable, not contaminated, and at the desired density is required throughout all protocols.

Adherent Cell Stimulation and Collection

Cell stimulation and collection will be performed in a tissue culture laboratory/Class II Biological Cabinet.

Treatment Stimulation

1. Prior to stimulating your cells, prepare the appropriate lysis buffer.
2. Prepare the stimulation media immediately before use and prewarm to 37°C. Use 10 mL of media/150 mm dish for up to 2 hours stimulation time. For stimulation time >2 hours, use 20 mLs of media/150 mm dish.
3. Using suction and a Pasteur pipette, remove the culture media from the dish or flask and discard it, following your institute’s waste disposal protocol.
   Note: In some instances, cells may need to be washed to remove serum components.
4. Add the stimulation media.
5. Return the plates to the 37°C, 5% CO2 incubator for the specified time.

Cell Collection-Strongly Adherent Cells

1. Using suction and a Pasteur pipette, remove the stimulation/culture, media from the dish or flask and discard it, following your institute’s waste disposal protocol.
2. Wash the plates 2 times with ice cold PBS. Rock the plates several times, using at least 10 mL/150 mm dish. Remove each wash as in Step 3 above, or refer to the “Cell Collection - Weakly Adherent Cells” section below, if cells are detaching in PBS.
   • - It is essential that all PBS be aspirated to prevent unwanted dilution of the final product.
   • - Insufficient washing will contaminate lysate with media components (especially serum/BSA), may erroneously elevate protein concentration, and will affect performance in immunoblotting.
3. Proceed with the appropriate lysate protocol (refer to “Native Lysates” protocols).

Cell Collection-Weakly Adherent Cells

Since some stimulation conditions can weaken the cell’s attachment to the plate, it is necessary to modify the cell collection technique if this is suspected. This can happen with colcemid treatment and infected/transfected cells.

Method

1. Scrape cells and media into centrifuge tubes.
2. Centrifuge at 1000 rpm for 10 minutes.
3. Wash 2 times with 10 mLs of ice cold PBS, centrifuging in between washes as in Step 2.
4. Proceed with the appropriate lysate protocol, adding lysis buffer immediately after washing (refer to “Native Lysates” protocols).
   1. Use the guide for adherent cells for the amount of lysis buffer to be added.
   2. The cascade of events prompted by stimulation may continue even though the stimulation media is removed and the plates or cells are washed.
   3. Proceed with the appropriate lysate protocol (refer to “Native Lysates” protocols).

Adherent Cell Lysate Protcol Summary

1. Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS.
2. Add ice-cold modified RIPA buffer to cells (1 mL per 107 cells/100 mm dish/150 cm2 flask; 0.5 mL per 5 x 106 cells/60 mm dish/75 cm2 flask).
3. Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge tube. Gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells.
4. Centrifuge the lysate at 14,000 xg in a precooled centrifuge for 15 minutes. Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet. Dilute the cell lysate at least 1:10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent. At this step, the sample can be divided into aliquots and stored at -20°C for several months.

Non-Adherent Cell Stimulation and Collection

Treatment/Stimulation

Cell stimulation and collection will be performed in a tissue culture laboratory/Class II Biological Cabinet.

1. Prepare stimulation media immediately before use and prewarm to 37°C.
   • If a large volume of cells is involved, cells may need to be concentrated to a lesser volume of media so that the stimulating reagent is not wasted.
   • Treatments can be added directly to the cell culture plates/flasks if they contain the appropriate basal media (i.e., if serum free media is not required).
2. If the volume of the cells needs to be reduced or the media changed, i.e. to serum free or serum containing media, collect the cells in an appropriate centrifuge tube and spin at 1000 rpm for 10 minutes at room temperature (RT). Aspirate the supernatant, wash if necessary to remove serum components, and resuspend the pellet in the pre-warmed stimulation media.
3. Incubate at 37°C for the specified time.

Cell Collection

1. Calculate the amount of lysis buffer needed.
2. Prepare the lysis buffer.
3. Pool the cells into a new disposable centrifuge tube.
4. Using 10 mLs of ice cold PBS, wash the plate or flask, remove the PBS and use it to sequentially wash the remaining plates or flasks. After washing the last plate or flask, add the wash to the cell pool. This assures that no cells are left behind.
5. Remove a 100 μl aliquot of the cell suspension and save it for a cell count. Record the total volume of cell suspension collected.
6. Centrifuge at 1000 rpm for 10 minutes.
7. During centrifugation, count the cells and calculate the total cell number, i.e., the total volume of cell suspension collected (mLs) x viable cells/mL.
8. Resuspend the cell pellet with ice cold PBS in 10-25% starting volume.
9. Centrifuge the cells as in Step 6, and aspirate the supernatant.
10. Proceed with the appropriate lysate protocol (refer to “Lysate Preparation” protocols).

Lysate Preparation-Native Conditions

Native Lysates-Modified RIPA

Cells are generally lysed by treating with detergents, sonication or by repeated freeze and thaw cycles. Extraction conditions release proteases from the cells, thus the need for protease inhibitors in the lysis buffer. Some protease inhibitors, for example, PMSF and benzamidine, have a half-life of only 30 minutes. Add just prior to use.

1. 1. Modified RIPA Buffer Calculation
   1. Adherent cells: use 2 to 3 mL ice cold modified RIPA buffer per each 150 mm plate depending on cell confluency (i.e., cell density, 2 mL for 80% confluency to 3 mL for a 100% confluent monolayer).
   2. Non-adherent cells: use 1 mL ice cold modified RIPA buffer for each 107 cells. For example: if there are 8x107 total cells, 1x8 mLs of RIPA, or 8 mLs is needed.
2. 2. Lysate Processing
   The following steps should be carried out in a cold room on ice.
   1. Add the appropriate amount of lysis buffer to washed cells (refer to section 3a).
   2. Adherent cells: Tilt the plate and use a cell scraper to pool the cells to one edge of the plate.
   3. Non-adherent cells or scraped adherent cells: Resuspend pellet in lysis buffer.
   4. Transfer the lysate to a new pre-chilled 50 mL centrifuge tube suitable for the SS-34 rotor.
   5. Gently rock the lysate on an orbital shaker in the cold room for 15 minutes.

Kinase Assay Lysate Preparation

Native Lysates-IPK Lysis Buffer/Buffer A

It is not possible to accurately measure activity of a specific kinase in most cell lysates because of the presence of other kinases that phosphorylate the same substrates, thereby interfering with the assays. Thus, it is critical to immunoprecipitate proteins with kinase activity in order to study signal transduction pathways. After immunoprecipitation, kinase activity is measured in one of two ways. It may be accomplished by subtracting the kinase activity in unstimulated cell lysates from that measured in stimulated cell lysates, or by comparing the kinase activity of the precipitate of the test antibody in stimulated cell lysates with the activity of the precipitate of a corresponding nonspecific antibody (i.e. rabbit or sheep polyclonal IgG/antisera) and/or preimmune sera.

Setup

Typically, 500 to 1,000 :g of lysate is used per assay point. An assay performed in triplicate, with controls, uses a large quantity of cell lysate. Different cell lines require the use of growth factors, or stimulants, at different time points to activate the kinase of interest. Kinases may be inactivated by proteases or phosphatases present in cell extracts. Once the conditions for the specific cell line are met, it is very critical that the correct cell lysis buffer be used to prevent this inactivation.

Buffer Calculation

Refer to the Appendix for formulation.
Adherent cells: Add 1 mL of ice cold cell lysis Buffer A/150 mm plate.

Lysate Processing

The following steps should be carried out in the cold room on ice.

1. Pre-chill 1.5 mL Eppendorf tubes at -20°C.
2. Add the appropriate amount of cell lysis buffer (refer to the NRSB-MINUS Calculation under “Denatured Lysates” below).
3. Tilt the plate and use a cell scraper to pool the cells to one edge of the plate.
4. Immediately remove the cell lysis buffer containing the cell extract from the dishes and place in an ice cold conical tube.
5. Mix thoroughly and aliquot 0.5 mL into pre-chilled Eppendorf microcentrifuge tubes.
6. Immediately place into a chilled cryorack and submerge in liquid nitrogen for 2 minutes. Remove and place immediately in labeled box; store at -70°C.
7. Remove a sample, thaw and clarify by centrifuging at 14,000 rpm for 2 minutes at 4°C Remove the supernatant, and quantitate using a Bradford Protein Microassay for RIPA cell lysates.
8. After thawing, centrifuge for 2 minutes, 14,000 rpm at 4°C before using for testing purposes. This is necessary to remove any bulk precipitate.

Lysate Preparation-Denatured Conditions

Lysing cells with reducing sample buffer (Lammeli) lacking Bromophenol blue has several advantages. It is quick, simple and reduces concerns that the protein of interest has not been solubilized. The presence of 2% SDS ensures total lysis of the nucleus, and results in the extraction of genomic DNA, which should be sheared by sonication to reduce viscosity. The absence of bromophenol blue enables protein quantitation via the Bradford assay. This lysis procedure is denaturing, and therefore may not be suitable for immunoprecipitation experiments in which the immunoprecipitating antibody recognizes secondary or tertiary protein structure.

Non-reducing Sample Buffer (NRSB-Minus) Calcuation

For Adherent cells: use 2 to 3 mL of NRSB-MINUS (refer to appendix) per each 150 mm plate depending on cell confluency (i.e., cell density, 2 mL for 80% confluency to 3 mL for a 100% confluent monolayer). Two to 3 mLs of NRSB-MINUS are necessary to sufficiently extract nuclear proteins.

Lysate Processing - Process in a Fume Hood

1. Bring NRSB-MINUS to a boil.
2. Add the appropriate amount of boiling NRSBMINUS to the washed cells (refer to the NRSBMINUS Calculation section above) and swirl to distribute the lysis buffer over the entire surface of the plate.
3. Adherent cells: Tilt the plate and use a cell scraper to pool the cells to one edge of the plate.
4. Transfer lysate to an appropriate sized tube and boil for an additional 10 minutes.
   1. Depending on the volume of lysate, use either microcentrifuge tubes or 15 mL conical tubes
5. Shear DNA by putting the lysate through a 26 gauge needle or sonicate the lysate.
   1. Minimize foaming.
   2. Pass the lysate through a 26 gauge needle until the sample is no longer viscous.
   3. Sonicate by performing 4 cycles at 30 sec/cycle or until the sample is no longer viscous.
      1. Sonication conditions for the Branson sonifier 450 cup sonicator:
         • Sufficient water added to the cup to cover the material in the sample tube.
         • Duty cycle set at 80.
         • Output set at 10.
      2. Both sonication and passage through a 26 gauge needle help reduce the viscosity of the sample by fragmenting nuclear DNA.
   4. If the lysate is still viscous after sonication, passage through a 26 gauge needle to complete fragmentation of nuclear DNA.
6. Centrifuge for 10 minutes in a minifuge at 1000 rpm. For larger volumes, spin at 15,000 xg for 15 minutes at room temperature. Do not spin at 4°C
   1. Centrifuging at 4°C will result in precipitation of SDS.
   2. Centrifugation helps remove insoluble material.
7. Save the supernatant and discard the pellet following your institute’s waste disposal protocol.
8. Determine the lysate concentration using the Bradford Protein Assay for RIPA Lysates Microassay.
9. Dilute to 1.1 mg/mL using NRSB-MINUS.
10. Add 1/10th the volume of 0.5% Bromophenol Blue (lysate is now at 1 mg/mL) and aliquot 650 mL per microcentrifuge tube.
    1. To 5 mLs of lysate at 1.1 mg/mL you would add 0.5 mLs of 0.5% Bromophenol blue.
11. Store the lysate at -70°C. Shelf life is 6 months.

Cell Lysate Extracts-Hela Nuclear Lysate

HeLa cells, >109 cells from either spinner culture or 150 mm plates (~40 X 150 mm plates).

Method

1. Pellet the HeLa cells by centrifugation for 10 minutes at 2000 rpm. The remainder of the procedure must be performed on ice.
2. Resuspend the cells in 5 packed cell pellet volumes of ice cold PBS.
3. Pellet the cells for ten minutes at 2,000 rpm.
4. Resuspend the pellet in 5 packed cell pellet volumes of buffer #1 and incubate for 10 minutes on ice.
5. Pellet the cells for 10 minutes at 2,000 rpm.
6. Resuspend the pellet in 2 packed cell pellet volumes of buffer #1 and lyse with 10 strokes of Dounce homogenizer (B type pestle).
7. Centrifuge the homogenate for 10 minutes at 2,000 rpm to pellet nuclei.
8. Carefully remove the supernatant. Keep the nuclear pellet.
9. Recentrifuge the nuclear pellet for 20 minutes at 25,000 xg to remove residual cytoplasmic material.
10. Resuspend the crude nuclei (pellet) in 3 mL buffer 2/109 cells using 10 strokes with a Dounce homogenizer (B type pestle).
11. Gently stir the resulting suspension with a magnetic stirring for 30 minutes.
12. Centrifuge for 30 minutes at 25,000 xg.
13. Dialyze the clear supernatant against 50 volumes of buffer #3 for 5 hours (overnight is acceptable).
14. Centrifuge the dialysate at 25,000 xg for 20 minutes. Carefully remove supernatant for quantitation. Package and freeze at -70°C.

Buffers

Buffer #1
HEPES	10mM
MgCI2	1.5mM
KCl 1	0 mM
DTT*	0.5 mM
Buffer #2
HEPES	20 mM
Glycerol	25 %
NaCl	0.42 M
MgCl2	1.5 mM
EDTA	0.2 mM
PMSF*	0.5 mM
DTT*	0.5 mM
Buffer #3
HEPES	20 mM
Glycerol	20 %
KCI	0.1 M
EDTA	0.2 mM
PMSF*	0.5 mM
DTT*	0.5 mM

Membrane, Cytosol and Nuclear Preparations

1. Weigh out organ and cut it into cubes.
2. Add ice-cold RIPA buffer containing 1 mM DTT and inhibitors, but not NP-40 or sodium deoxycholate. Add 1L of buffer per 250 mg of tissue.
3. Grind the material in a blender on low power using 30-second bursts until fully homogenized. Allow the material to cool for 1 minute between bursts.
4. Pour the material into pre-cooled JA14 centrifuge tubes, balance, and centrifuge.
5. Centrifuge nuclei and unbroken cells at 2,900 xg for 20 minutes.
6. Pour off supernatant (which contains cytosol and membrane fractions) into another pre-cooled tube and set it aside on ice. Save the nuclear pellet.
7. Resuspend the nuclear pellet saved in step 6 in 10 mL of RIPA buffer containing detergents. Check the protein concentration and dilute it with PBS if necessary.
8. Spin down membranes by centrifuging the supernatant from Step 6 at 29,000 xg for 45 minutes.
9. Pour off the supernatant from Step 8, save as cytosolic fraction, and process it as described in Step 12. Process the remaining pellet as in described in Step 10.
10. Resuspend the pellet from Step 9 in 10 mL of RIPA buffer containing detergents.
11. Centrifuge the resuspended membranes from Step 10 at 15,000 xg for 20 minutes. Save the supernatant as the membrane fraction. Quantitate and dilute as needed.
12. To the supernatant from Step 9, concentrate the preparation by adding glacial acetic acid, drop by drop, until pH reaches 4.5.
Caution: glacial acetic acid is caustic. Perform with caution in a hood using gloves, goggles, and a lab coat.
13. Centrifuge the concentrated cytosol from Step 12 at 15,000 xg for 20 minutes.
14. Discard the supernatant, and resuspend the pellet in 10 mLs of PBS. Quantitate and dilute with PBS as needed.

Dephosphorylation Using Lamda Phosphatase

Lysate Sample Dephosphorylation

Method

1. To a microcentrifuge tube, add 80 μg of lysate. The amount may be scaled up or down as needed.
2. Add 6 μl of l-PPase Buffer. Final concentration: 50 mM Tris, pH 7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 2 mM MnCl2.
3. Add 400 units of l-Phosphatase (Cat. No.14-405).
4. Incubate at 30°C for 20 minutes.
5. The protein sample or lysate is now ready for analysis or further preparation, depending on the application.

Nitrocellulose Dephosphorylation

Method

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.
2. Block the blotted nitrocellulose in freshly prepared TBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hour at 20-25°C with constant agitation.
3. Incubate the nitrocellulose in TBS containing 1% bovine serum albumin (BSA), 0.1% Triton X-100 and 2 mM MnCl2, and where dephosphorylation of proteins is desirable, 400 U/mL l-phosphatase (Cat. No. 14-405). Incubate for 4 hours at room temperature, or overnight at 4°C.
4. After incubation, wash the nitrocellulose in PBS-0.1% Tween 20 for 3 to 5 minutes.
5. Rinse the nitrocellulose in 4 to 5 changes of water and continue on with Western blotting protocol.