Making Poly-L-proline Columns for Profilin Purification
Making a poly-L-proline Sepharose column for purification of profilin
N.B. This protocol makes a lifetime supply of pLp resin.
Cyanogen bromide, Sigma Aldrich Chemicals. Store tightly sealed at 4 C.
Sepharose CL4B, Pharmacia or Sigma.
Acetonitrile, reagent grade from Sigma.
Poly-L-proline, Sigma catalog #p-2254.
Magnetic stir plate, stir bar, balance and pH meter – all set up in fume hood.
Whatman 541 filter paper circles.
2 M Na2CO3
2 M NaOH
10X Quenching buffer (bead buffer)
1 M NaCl
1 M glycine
100 mM Tris pH 7.5
Storage buffer (1X)
10 mM Tris pH 7.5
50 mM KCl
1 mM EDTA
0.002% Sodium Azide
Activation of Sepharose CL4B
Take approximately 100 ml Sepharose CL4B and wash with 2 L ddH2O in Buchner funnel or sintered glass funnel fitted with Whatman 541 paper circle.
Remove excess water so that resin becomes a nice damp cake.
Place resin into 1 L glass beaker and add 100 ml ddH2O (make 50% resin slurry).
Add 200 ml of 2M Na2CO3 to resin (1 M final conc. of Na2CO3) and start stirring on stir plate.
In a fume hood, weigh out 25 gm cyanogen bromide and mix with 50 ml acetonitrile.
Add CnBr:acetonitrile to vigorously stirring resin slurry.
Stir for 2 minutes (in fume hood) at room temperature. Time with stop watch.
Transfer slurry to Buchner funnel fitted with Whatman 541 paper circle. In fume hood, remove excess liquid and wash with 6 liters of ddH2O.
Resuspend to 200 ml final volume in ddH2O and add 0.05 volumes of 2M Na2CO3 (final conc. 0.1 M).
Coupling Poly-L-proline to resin
Dissolve 1 gm of pLp in 80 ml ice-cold ddH2O.
Add pLp solution to 50% slurry of activated Sepharose with stirring.
Stir for 2 hours at room temperature.
Transfer to cold room and stir overnight at 4 C.
Quench reaction by adding 0.1 volume of 10X bead buffer.
Wash resin in Buchner funnel with 2L ddH2O and store in 1X storage buffer.