• Pepstatin (Sigma Chemical Co.)
• Sodium orthovanadate (Na3VO^): prepare a small amount of a 0.1-0.5 M stock solution in water immediately prior to use
• Buffer C: 20% (v/v) glycerol, 20 mM Hepes pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA
• Freezing medium; 50 mM Tris pH 8,3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA
• Trypan blue (0.4% solution) for vital dye staining (Life Technologies)
• Serum-free RPM! 1640 tissue culture medium
1 Plate an appropriate number of the macrophages in 60 mm tissue culture dishes to create a confluent monolayer of adherent cells at the initiation of the experiment.8
2 Serum starve the cell cultures for 24-72 h at 37°C once they reach confluency."
3 Stimulate the serum-starved macrophages as required for the experiment underway.
4 Remove medium from the monolayers and spin in a centrifuge for 5-10 min, 4°C, 250-500 g to collect any cells growing in suspension.
5 Add 4 ml ice-cold 1 x PBS to the remaining monolayers and harvest the adherent macrophages by an appropriate method (see Chapters 1 and 2).
6 Resuspend the pelleted suspension cells using the same 4 ml wash fluid and collect all the cells by centrifugation as in step 4.
7 Resuspend the cell pellets in 2-5 ml of NE buffer.
8 Incubate the cells on ice for 5 min.
9 Collect the cells by centrifugation as in step 4.
10 Resuspend the cell pellets in 800 (j.1 of NE buffer supplemented to 1 mM with DTT, 0,4 mM with PMSF, and 100 |aM with Na3V04.
11 Lyse the resuspended macrophages by passing the cells repeatedly through a 25-gauge needle attached to a 1 ml syringe. Continue to homogenize the cells until the nuclei are released from the cytoplasm of lysed cells, as monitored by staining with trypan blue.c
12 Collect the released nuclei by spinning at 12000-15 000 r.p.m. at 4X for 10 sec in a microcentrifuge.
13 Remove and discard the supernatant fluid.
14 Centrifuge the nuclei once more for 2 min under the same conditions as in step 12, using 1 ml of NE buffer.'
15 Remove the supernatant fluid and resuspend the nuclear pellet rapidly in three pellet volumes of buffer C supplemented to 3.6 jxg/ml with aprotinln, 1.2 jig/ml with pep-statin, and 1.2 ixg/ml with leupeptin, and with DTT, PMSF, and Na3V04 as in step 10."'
16 Incubate the suspension on ice for 20-30 min,
17 Centrifuge the mixture for 3 min as in step 12 and recover the supernatant fluid.
18 Determine the protein concentration of the extracts.
19 Store the supernatant fluid in 25-50 p.1 aliquots at -70°C.e
-1 Expand cell cultures (for macrophage cell lines) or purify sufficient primary macrophages to ensure that the cell number at the start of the extraction will be at least 5 x 107 total cells. bThe period of serum starvation should be sufficient to render the macrophage population quiescent, but should not have a deleterious effect on cellular morphology or viability. c Nuclei should be as close to 100% free of any cellular debris as possible, as visualized by trypan blue staining.
d Alternatively, the nuclear pellet can be resuspended in 100 p.1 of freezing buffer at this point and stored in liquid nitrogen for use in nuclear run-on experiments (see Protocol 2). * Nuclear extracts are stable for four to six weeks under these conditions.
fkation of nuclear DNA binding factors. Protocols 8 and 9 first detail methods for preparation of the two critical components of EMSA analysis, namely the nuclear extracts and the radiolabeled oligonucleotide probes. Protocol 10 then describes the use of these reagents for I;MSA studies. Information concerning additional methodologies for identification of DNA binding proteins is not provided here, but can be obtained from more extensive methodology texts (12).
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