This protocol is for a gel mobility shift assay for RPA binding to labeled dT30*

• Assemble a cocktail (5 ul/rxn) which contains Filter Binding Buffer, BSA and end-labeled DNA (dT30*) on ice (make up several extra rxn volumes to allow for pipetting error and count the reaction mix in order to precisely calculate the amount of DNA added to each reaction)
• Add the appropriate amount of protein into separate tubes for each reaction on ice and adjust volume to 10 µl with HI-30 or another low salt buffer (maintain equal amounts of salt in each reaction)
• Aliquot 5 µl of cocktail containing DNA into each tube on ice either before or after the protein is added to the reaction
• Note: if adding more than one protein, pre-incubate proteins together for 5 minutes at 25°C
• Total reaction volume is 15 ul

Below is a sample titration of RPA on dT30* over three orders of magnitude with two points per magnitude (we use log spacing for protein concentrations).  Note we usually use 0.2-2 fmoles of DNA fragment.  0.2 fmoles is preferred to give equilibrium binding conditions for RPA. 

Cocktail (3X)                                   

Stock                  Solution                  Volume (µl)                  Final/Rxn

10X                       FBB                            15                               1X

20 µg/µl                 BSA                           0.38                        50 ng/ul

10 fmol/µl             dT30*                         2.00                          2 fmol

                              ddH20                         32.6                 

                               Total                            50                                   

Simple titration of RPA

Rxn                Cocktail                  HI-30                  RPA                  RPA                  Total

Number             (µl)                        (µl)                  (fmol)                  (µl)                  Volume (µl)

1                           5                            10                        -                         -                          15

2                           5                              9                  1.00                       1 (a)                       15

3                           5                            6.84                  3.16                    3.16 (a)                  15

4                           5                              9                  10.0                       1 (b)                       15

5                           5                           6.84                  31.6                     3.16 (b)                  15

6                           5                             9                   100                        1 (c)                       15

7                           5                          6.84                 316                        3.16 (c)                  15

RPA concentrations (diluted in HI-30)

   (a)  1 fmol/µl

   (b)  10 fmol/µl

   (c)  100 fmol/µl

dT30*:  concentrations determined with each labeling

• Incubate reaction for 20 minutes at 25°C (water bath)
• Add 1.5 ul of 10X Retardation Buffer (loading buffer) to reaction
• Load samples onto a 1% Agarose gel in 0.1X TAE with 0.1X TAE running buffer
• Note:  for sharper bands use a 1 mm comb
• Run dye front approximately halfway down the gel (100-120 volts, ~1.5 hr on big gels with 2 combs)
• free DNA runs ahead of the dye front and the shifted band runs behind the dye front
• Rinse gel in water for several minutes
• Dry down gel on DE81 paper on top of two pieces of Whatman paper
• Expose dried gel to film overnight

10X Filter Binding Buffer (10X FBB)

300 mM HEPES (pH 7.5)

1 M NaCl

50 mM MgCl2

1 M NaCl

5% Inositol

10 mM DTT

10X Retardation Buffer

40% Glycerol

0.04% Bromophenol Blue