Molecular Biology Lab 312L

Outline: Mobility Shift Assay

1. Prepare serial dilutions of IHF protein in TE buffer. Keep stock and dilutions on ice. This is important! The stock IHF is 0.7 mg/ml.

Add the correct amount of TE buffer to all of the dilution tubes. Then add the stock IHF protein to the first dilution tube (1:4). Mix; transfer diluted protein to the next tube (1:8). Mix and repeat.

1:2 dilution = 4 ml stock IHF into 4 ml TE
1:4 dilution = 3 ml stock IHF into 9 ml TE
1:8 dilution = 6 ml of 1:4 dilution into 6 ml TE
1:16 dilution = 6 ml of 1:8 dilution into 6 ml TE
1:32 dilution = 4 ml of 1:16 dilution into 4 ml TE
1:48 dilution = 4 ml of 1:16 dilution into 8 ml TE
1:64 dilution = 4 ml of 1:32 dilution into 4 ml TE

2. Assemble DNA-protein complexes with Lambda attP and IHF. Prepare complexes at room temperature.

sample	attP fragment	IHF protein
1	9 ml	1 ml of TE buffer (no protein)
2	9 ml	1 ml of 1:64 dilution
3	9 ml	1 ml of 1:48 dilution
4	9 ml	1 ml of 1:32 dilution
5	9 ml	1 ml of 1:16 dilution
6	9 ml	1 ml of 1:8 dilution
7	9 ml	1 ml of 1:4 dilution
8	9 ml	1 ml of 1:2 dilution

3. Add 2 ml of 6x Tracking Dye to all of the reaction tubes. Load on the acrylamide gel starting with sample #1 (no protein) on the left. A ladder lane is optional. Run the gel at 70 volts.