Gel mobility change analysis experiment

Summary

In this experiment, we used the same complementary oligonucleotide used for DNA affinity chromatography (see the end of the Introduction to Experiment 3 for the exact sequence) to perform the gel mobility shift analysis of AP-1. This experiment was derived from the Guide to Protein Purification and Characterization by Houzhu Zhu.

Operation method

Gel mobility change analysis experiment

Materials and Instruments

Acrylamide Bisacrylamide Ammonium persulfate TEMED (N N N' N' tetramethylethylenediamine) Glycerol (50%;v v) Competitive DNA Mobility Shift Analysis Probes TBE Gel Mobility Shift Analysis Buffer TE

Move

makings

Acrylamide

Bisacrylamide

Ammonium persulfate (10%;w/v)

TEMED (N,N,N',N', tetramethylethylenediamine)

Glycerin (50%;v/v)

Competition DNA (soluble in TE) (type and amount of competition DNA can vary with protein type and purity; proteins purified by affinity chromatography do not use competition DNA)

Mobility shift analysis probe (made from complementary oligonucleotides phosphorylated by T4 polynucleotidase and [ ^γ-32P ]ATP)

Reagents

TBE (10X)

Gel Mobility Shift Analysis Buffer (10x)

TE

(For the recipe, see "Reagent Preparation", pp.131~138)

Operating Procedures

1) Prepare a 5% polyacrylamide gel (20 cmx20 cmx1.5 mm) according to the following recipe.

Acrylamide (30%; w/v)/bisacrylamide (0.8%; w/v) 16.2 ml

TBE(1x) 5.0 ml

_H20 78.8 ml

Mixed, filtered through a 0.45um membrane, then 600ul of 10% (w/v) ammonium persulfate and 48ul of TEMED were added.

2) Gel polymerization for 30~60 min, pre-electrophoresis at 100V for 30 min before sampling.

3) For each sample, mix the following ingredients in a reaction tube on ice:

Mobility shift analysis buffer (10x) 1ul

Glycerol (50%; v/v) 2ul

Competition DNA (add as needed; for pure proteins, add 1ul of _H20 instead) 1ul

Protein fraction (dissolved in buffer Z containing 0.1mol/LKCl) 5ul

Mobility shift analysis probe (~20000cpm/ul) 1ul

Total volume: 10ul

Flick the reaction tube for gentle mixing.

4) Incubate the sample at room temperature for 10~15 min.

5) Add the sample directly to the gel. It is not recommended to add dyes to the sample because dyes often inhibit the binding of proteins to DNA.

6) Electrophoresis at 100V at room temperature until the reference dye bromophenol blue (spotted on the protein-free lane of the plate) migrates about 10 cm below the plate.

7) The gel is dried and then autoradiographed (typically, -80°C for several hours or overnight).

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