Gel Shift Protocol

Electrophoretic Mobility Shift Assay with extract:

1µl of 1mM spermidine (0.1mM final concentration)

1µl of RNA (50-100k)

4µl of extract

0.5µl of 25mM EDTA

4µl of Buffer D

Incubate at 30°C for 5 minutes

Add 1µl of heparin (40 mg/ml)

Ice for 5 minutes

Add 1µl 10x urea free loading buffer

Run on pre-run 5% non-denaturing gel at 200 V for 2 hours

With recombinant Protein:

1µl protein

7µl Buffer D

Buffer D:

20mM Hepes-KOH
20% Glycerol
100mM of KCl
0.2mM EDTA
1mM DTT

10x Urea Free Loading Buffer:

30% Glycerol
0.5% Bromophenol Blue
0.5% Xylene Cyanol

5% Non-Denaturing Gel:

10ml of acrylamide
6ml of 10x TBE
600µl of 10% APS
45µl of temed
45.35ml of H₂O