DESARROLLO DE LA PROPUESTA

The gel shift or electrophoretic shift assay provides a simple and rapid

method of detecting DNA-binding proteins. This method has been widely used

in the study of sequence-specific DNA-binding proteins, such as transcription

factors (Briggs et al. 1986; Lee et al. 1987). The assay is based on the

observation that complexes of protein and DNA migrate through a

nondenaturing polyacrylamide gel more slowly than free DNA fragments or

double-stranded oligonucleotides. The gel shift assay is performed by incubating

a purified protein or a complex mixture of proteins (such as cell or nuclear

extracts) with end-labelled DNA fragment containing the putative protein-

binding site. The reaction products are then analysed on a nondenaturing

polyacrylamide gel and exposed to X-ray film. The specificity of the DNA-

binding protein for the putative binding site is established by competition

experiments using non-radioactive DNA fragments or oligonucleotides

containing a binding site for the protein of interest, or other unrelated DNA

Reagents: Binding Buffer: 10 mM HEPES pH 7.9 50 mM KCl 0.2 mM EDTA 2.5 mM DTT 10 % Glycerol 0.05% NP-40

Protease inhibitor cocktail

For 100 ml

0.238 g HEPES

0.373 g KCl

0.1 ml 200 mM EDTA stock solution

10 ml Glycerol

50 pi NP-40

Make up to 100 ml autoclave and store at 4 ^C

Add DTT and protease inhibitors immediately prior to use. 1:100 (250 mM stock

DTT).

0.386 g DTT per 10 ml H2O, freeze aliquots at -40 ^C

Gel loading buffer (10 X):

250 mM Tris-HCl pH 7.5

0.2 % Bromophenol blue

For 100 ml : 3.94 g Tris 0.2 g Bromophenol blue 40 ml Glycerol Running Buffer (5 X) pH 8.3 25 mM Tris Base 190 mM Glycine For 2000 ml : 3.028 g TRIS 28.527 g glycine

Dilute 1:5 for working buffer: 400 ml 5 x buffer in 1600 ml H2O.

Method:

DNA binding reactions and electrophoretic mobility shift assay (EMSA)

Protein concentrations in nuclear extracts were measured using the

method of Bradford (Bradford 1976) and 5 pg of extract used in binding

reactions. A double stranded NF-kB binding consensus oligonucleotide, 5'-A G T TGA GGG GAG TTTCCCAGG C-3’ (Promega, Southampton, UK) was end labelled

with ^^P using Ready-To-Go T4 polynucleotide kinase (Amersham

Biosciences, Bucks, UK). Unincorporated nucleotides were removed using

sephadex G-25 microspin columns (Amersham Biosciences).

Binding reactions were performed with 127 finol of ^^P labelled

oligonucleotide for 30 min at room temperature in 10 mM HEPES (pH 7.9): 50

mM KCl, 0.2 mM EDTA, 2.5 mM DTT and 10 % glycerol and 1 pg of poly (dl-

dC). Protein-DNA complexes were resolved on 7 % native polyacrylamide gels

stimulated HeLa cell nuclear extract (Promega) was used as a positive control

and band specificity was confirmed by the addition of 30-fold excess of cold

specific oligonucleotide.

End-labelling of DNA fragments for use in band-shift assays.

T4 Polynucleotide Kinase (PNK) is a tetrameric protein composed of

four identical subunits. It is widely used in molecular biology to radiolabel

nucleic acids and to add a phosphate to the 5'-end o f dephosphorylated nucleic

acids to facilitate ligations.

Ready-To-Go T4 Polynucleotide Kinase is a single-dose, ambient-

temperature stable format of the enzyme. 25 pi of water was added to the tube

containing the Ready-To-Go T4 PNK. The tube of reconstituted Ready-To-Go

T4 PNK was incubated at room temperature for 2 to 5 minutes, then the contents

of the tube were mixed by gentle pipetting up and down. 5-10 pmol of 5'-ends

of oligonucleotide and sufficient water was added to bring the reaction volume

to 49 pl.l pi of (gamma-32P) ATP (3000 Ci/mmol, 10 pCi/pl) was added and

mixed gently, then centrifuged briefly to collect the contents at the bottom of

the tube. The tube was incubated 30 minutes at 37°C. The reaction was stopped

by adding 5 pi o f 250 mM EDTA.

Note: Using more or less than 50 pi to reconstitute the reaction mix will result in

less than optimal buffer concentrations.

Removal of unincorporated using Sephadex microspin G-25 spin columns

(Apbiotech product no: 27-5325-01)

To remove the free or unbound from the labelled reaction mixture, the

solution was allowed to pass through a column containing resin. The resin was

resuspended in the column by vortexing. The top cap was removed and snapped

off the bottom closure and both were discarded. The column was placed in a

1.5 ml screw-cap microcentrifuge tube for support. Alternatively, the cap was

cut from a flip-top tube and this tube was used as a support. The column was

placed in a variable speed microcentrifuge. A digital timer and the

microcentrifuge were started simultaneously.

"Pulse spin" should not be used as this will override the variable speed setting.

The column was spun for 1 minute at 735 x g.

The column was transferred to a new 1.5 ml tube and the sample was

slowly applied to the centre of the angled surface of the compacted resin bed

without disturbing the resin. Any flowing samples around the sides of the bed

should be avoided.

The column was spun for 2 minutes at 735 x g. The purified sample was

collected in the bottom of the support tube. The column was discarded into a

radioactive waste store.