Analysis of samples bv electrophoretic mobility shift assays (EMSA)

Materials

• Diisopropyl fluorophosphate (DIFP) (Sigma) • [y-32p] ATP (Amersham International)

• T4 polynucleotide kinase and buffer (Promega) • Acrylamide/Bis 30:0.8 30% (Boehringer, Mannheim)

Trichloroacetic acid (TCA) (Sigma)

Protein assay concentrate (BioRad, CA, USA)

Oligonucleotides M GF, IRF-1 (Oswel DNA Service, Edinburgh, UK), O ct 1 (Santa Cruz Biotechnology, Inc. CA, USA)

lOx Tris Borate EDTA (TBE) buffer, pH 8.35:- 108.9 g/L Tris, 55.7 g/L boric acid, 7.4 g/L disodium EDTA. Diluted 1:40 in distilled water for use.

Loading marker:- 80% (vol/vol) formamide, 0.1% (vol/vol) xylene cyanol, 0.1% (vol/vol) bromophenol blue, in 2 mM EDTA

W hole cell extract (WCE) buffer:- 20 mM HEPES pH 7.9, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA.

Nuclear extract (NE) buffer 1:- 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 m M EG TA .

NE buffer 2:- 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA.

Inhibitors:- 1 mM DTT, 50 mM Na ortho vanadate, 1 mM p glycerophosphate, 1 mM PMSF, 10 pg/m l aprotinin, 10 pg/ml leupeptin and 10 pg/ml pepstatin

Buffer A :- 10 mM Tris HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 5% (vol/vol) glycerol, 0.1% (vol/vol) NP-40, 1 mg/ml BSA

Buffer B:- 20 mM HEPES, pH 7.9, 50 mM KCl, 0.5 mM DTT, 1 mM EDTA, 8% (wt/vol) Ficoll

N on-denaturing 4% polyacrylam ide gel:- 125 ml distilled w ater, 20 ml acrylamide, 3.75 ml 10 x TBE, 1 ml 10% APS, 200 pi TEMED.

M ethods

2.7.1 Preparation of W hole Cell and Nuclear Extracts

Cells were washed and resuspended in binding buffer at a concentration o f 2x10^ cells/ml. 1x10^ cells were stimulated with or without GM-CSF, for 5 m inute at 37^C. The subsequent steps were carried out at 4^C. Cells were washed twice in PBS and incubated for 30 minutes with 1 mM DIFP in PBS. Cells were pelleted and whole cell or nuclear extracts were prepared. W CE were prepared using a method described by Scholer et al., (1989), cells were resuspended in 100 pi of W CE buffer containing inhibitors and snap frozen in ethanol/dry ice followed by rapid thawing in a 37®C w ater bath. After 3 cycles of freeze/thawing cell debris was rem oved by centrifugation at 12000 x g for 10 minutes at 4^C. Supernatants were then aliquoted, snap frozen and stored at -80^C. For NE a method described by Schrieber et a i ,

(1989) was used. All steps were perform ed at 4^C in the cold room. Cells were resuspended in 400 pi of NE buffer 1, plus inhibitors and incubated for 15 minutes, 15 pi of 10% NP-40 was added and the cells briefly vortexed. Nuclei were pelleted

by centrifugation at 12 000 x g for 5 minutes and the supernatants discarded. The nuclear pellets were resuspended in 50 pi of NE buffer 2 and rotated end-over-end for 20 minutes. Samples were then centrifuged for 5 minutes and the supernatants aliquoted, snap frozen and stored at -80^C.

2.7.2 Estimation of Protein Concentration

The protein concentration of each sample was m easured in duplicate using the technique developed by Bradford, (1976). The protein assay concentrate was diluted 1:5 (vol/vol) with distilled water and 1 pi of sample was mixed with 1 ml of diluted dye and allowed to stand for 5 minutes. The absorbance were m easured using a spectrophotom eter at 595 nM and the samples calibrated against a standard curve using bovine serum albumin ( 0, 1, 2, 5, 10, 15 pg/ml) standards.

2.7.3 Annealing and end-labelling oligonucleotide probes

Single stranded oligonucleotide probes, corresponding to the mammary gland factor (M G F ) b in d in g s e q u e n c e fro m th e b o v in e P c a s e in g e n e 5'-

AGATTTCTAGGAATTCAATCC-3', 3'-TCTAAAGATCCTTAAGTTAGG^' and

th e IR F -1 ( I n te r fe r o n R e s p o n s e F a c to r) b in d in g s e q u e n c e 5’-

GATCCATTTCCCCGAAATGA-3’, 3'-C TA G G TA A AG G G G CTT TAC T-5' w ere

annealed by heating to 95^C for 5 minutes followed by slow cooling to room temperature.

The double stranded probes corresponding to the MGF binding sequence and to the octam er binding m otif 5 -TG TCG A A TG CA A A TC A C TA G A A -3' from the H2 B

prom oter, w ere end-labelled by phosphorylation w ith [y-32P] A TP and T4 polynucleotide kinase (PNK). 25 ng of oligonucleotide was incubated for 30 minutes at 37^0 with 30 pCi of [y-^^P] ATP and 10 units of T4 PNK in PNK buffer. The reaction was stopped using 2 pi of 0.5M FDTA and 88 pi of distilled water. Any unincorporated radioactivity was removed by centrifugation at 1100 x g for 6 minutes through a G25 sephadex column. The activity of the labelled probe was m easured by trichloroacetic acid (TCA) precipitation. 2 pi of labelled probe was mixed with 800 pi distilled water, 10 pi FCS and 200 pi TCA and incubated at 4^C for 1 hour. The sample was then filtered onto 3mm W hatm ann paper and w ashed twice with 5% TCA followed by 70% ethanol. The filter paper was dried at 37^C for 10 minutes, placed into a scintillation vial with 4 ml of scintillation fluid and counted using a (3 counter.

2.7.4 EMSA

For binding reactions, 10 p.g of WCE or 5 |ig of NE were mixed with either buffer A for p casein and IRF-1 binding or buffer B for Oct binding and the following were added; 1 |Lig salmon sperm DNA, 0.5 pg Poly (dl-dC), 0.5 pM PMSF, 2 pg/m l aprotinin, 1 pg/ml pepstatin A, 1 pg/ml leupeptin in a final volume of 30 pi. The binding reaction mixture was incubated at 4^0 for 20 minutes before adding 250 pg of the relevant labelled probe, the reaction was allowed to continue for a further 15 m inutes at room temperature. The D NA-protein com plexes were separated by electrophoresis on a non-denaturing 4% polyacrylamide 0.25 x TBE gel (20 cm x 20 cm X 0.3 cm spacers) using a large gel apparatus (Protean II, BioRad, UK) The gels were pre-run at 4^C for 1 hour at a constant 150 V, before loading the samples and electrophoresing for a further 3 hours. A loading marker containing brom ophenol blue was run in the final lane to estimate the distance the samples had travelled since the free probe runs at a similar speed to the bromophenol blue. The gels were dried onto W hatman 3 mm paper and visualised by auto radiography.