1. (a) Suspension cells are collected by centrifugation from culture medium, (b) For adherent cells, remove the culture medium completely from the culture dish. Add 5 ml of PBS and shake gently to wash cells. Then remove PBS completely. Add 5 ml of ice-cold PBS containing PMSF and scrape off with the aid of a rubber policeman (do not trypsinize adherent cells). Transfer cells to a 15 ml centrifuge tube and centrifuge at 500 g for 5 min.
2. Remove the supernatant carefully from cell pellets and add 1.5 ml of ice-cold PBS/PMSF. Pipette cells gently to resuspend and transfer the cells to a 1.5 ml microcentrifuge tube.
3. Centrifuge at 500 g for 0.5-1 min with a swinging bucket rotor at 4°C.
4. Remove the supernatant carefully and add either TEN-T solution (described on p. 160) or RIPA buffer with protease inhibitors. Pipette to resuspend the cell pellet. The volume of lysis solution depends on the cell numbers harvested. We usually add 200 (d of TEN-T solution to 1 x 107 cultured cells.
5. Keep on ice for 15 min. Then centrifuge at 10000 g for 20 min. at 4°C (in the cold).
6. Recover supernatant and store at -80°C.
Tissue specimens are either frozen in liquid nitrogen (N2) and tissue powder is prepared by crushing in a mortar and pestle in N2 followed by solubilization with a lysis buffer, or fresh tissue pieces are mechanically homogenized and sonicated in lysis buffers.
1. Cut the tissue sample with scalpel blades into small pieces and transfer them to 1.5 ml Eppendorf tube.
2. Immediately add RIPAa lysis buffer to the tissue and homogenize using a motor-driven pestle (e.g. KONTES Scientific Glassware/Instruments, Vineland, NJb). The volume of lysis buffer depends on the volume of sample harvested. We usually add lysis buffer at a 5:1 ratio of buffer to tissue volume (5 vol. RIPA buffer:1 vol. tissue).
3. Incubate on ice for 15 min.
4. Centrifuge 800-1600 g for 20 min at 4°C to pellet debris.
5. Recover the supernatant and store at -80 °C.
' An alternative to RIPA solutions is to use a modified Laemmli solution. Resuspend tissues in boiling 1 x Laemmli buffer lacking bromophenol blue dye and 2-mercaptoethanol at a 5:1 ratio of buffer to tissue volume. Homogenize and centrifuge at 800-1600 g for 20 min at room temperature (SDS will precipitate at 4°C). Store the supernatant at -20°C. ''Alternatively, disrupt tissue using a probe sonicator. A 25 pim probe and cycles of 0.5 minutes each typically works well. (Avoid heating lysates).
Frozen tissue specimens may either be derived from tissue samples that were placed directly in liquid nitrogen (and stored in the same or at -80 °C) or derived from histological specimens which were frozen in OTC compound (outside tissue compound, Tissue Tek-II, USA, VWR) for cryosectioning. The latter typically involves submerging in cold isopentane which is sitting in a dry-ice-acetone bath. The frozen material is then placed into a pre-cooled plastic mould and OTC compound is poured over the tissue, thus encasing it in a 'plastic-like' covering. The samples are then stored at -80 °C and can be shipped on dry-ice.
If samples are embedded in OTC, this covering must first be removed. Place the tissue specimen in a petri dish that has been pre-cooled on dry-ice. Working quickly, use scalpel and forceps to remove the OTC coating.
1. Immediately submerge the tissue in liquid nitrogen in a pre-cooled mortar and pestle.
2. Grind the tissue to a fine powder and transfer with the remaining liquid N2 into a polypropylene centrifuge tube. (Caution. Do not close the tube completely or it will blow up! Close the cap partially to prevent tissue from bubbling out and hold in a partially closed position with a gloved finger until 'boiling' of the liquid N2 subsides.)
3. When the liquid nitrogen has nearly all evaporated, add 1-2 volumes (at least!) of RIPA buffer with protease inhibitors.
4. This will initially freeze. Let it thaw on ice.
5. Vortex vigorously.
6. Sonicate (e.g. Heat-Systems Ultrasonicator using the standard 1.5 mm diameter tip and 1-3 sec pulses and power settings of 2-4). If the sample heats beyond ~ room temperature, put on Ice and allow to cool. Sonicate until the solution is no longer viscous and has become clear, without residual chucks of tissue.
7. Centrifuge in a microcentrifuge (16000 g) for 20 min and transfer the supernatant to a fresh microcentrifuge (1.5 ml) tube.
8. Store the supernatant at -80°C or keep on Ice and proceed directly to protein quantification and subsequent SDS-PAGE/lmmunoblot analysis.
To quantify the total protein concentration in lysates, we use the bicinchoninic acid (BCA) protein assay reagent (PIERCE) for Triton X-100-solubilized RIPA lysates (11) and for Laemmli lysates, provided 0-mercaptoethanol has been withheld until ready to boil, and load samples in gels.
Use the Coomassie protein assay reagent (PIERCE) for Laemmli lysates because SDS interferes with the BCA method.
TEN-T solution. This solution is generally reliable for extraction of Bcl-2 family protein from most cultured cells. A modified version is useful for co-immunoprecipitation experiments in which 0.2-1 % (v/v) NP-40 is substituted for Triton X-100. Phosphatase inhibitors are optional, depending on whether experiments entail assessment of phosphorylation of Bcl-2 family proteins.
150 mM NaCl 1% Triton X-100 10 mM Tris (pH 7.4) 5 mM EDTA (pH 8.0)
Make from autoclaved solutions. Store at room temperature but add protease inhibitors fresh each time to the aliquot of the solution needed for your experiments.
RIPA solution. This lysis solution reliably extracts Bcl-2 family proteins for cultured cells and many tissues, including frozen tissues. It can be stored at room temperature and protease inhibitors are added immediately prior to use. Phosphatase inhibitors are optional, depending on whether the experiments entail assessment of phosphorylation of Bcl-2 family proteins.
1 % deoxycholate 0.1% SDS 5 mM EDTA
Laemmli solution. Ensures vigorous extraction of proteins from tissue specimens.
6.25 ml of 0.5 M Tris (pH 6.6) 11.25 ml of 10% SDS 5 ml of glycerol Bring volume to 45 ml Store at room temperature
When used as a loading solution for SDS-PAGE, the solution is typically prepared at 2 X concentration and mixed with an equal volume of sample lysate. In this case, BPB (bromophenol blue; 5 mg per 45 ml) is added and 10% 2-mercaptoethanol is included fresh each time to the portion needed. Protease inhibitors. Table 1 lists the protease inhibitors.
Phosphatase inhibitors (final concentrations). 10 mM sodium (^-glycerophosphate (pH 7.4)
1 mM Na3V04 (sodium orthovanadate) 5 mM NaF
2 mM Na4P207 (pyrophosphate) 50 mM 4-nitrophenyl phosphate 1 p,M microcystin-LR
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