Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 307

A. Covalent Coupling of Antibodies to Protein A Beads
Immunoprecipitation and tandem affinity purification both start with coupling the
antibody to Protein A resin. For antibodies generated in species with weak affinity to
protein A, protein G resin should be used. Coupling greatly reduces antibody
leaching during elutions. The tradeoff is a reduction in antibody efficacy because
coupling is performed using a bifunctional amine crosslinker that to a variable extent
will react with the antigen-binding site. The use of the crosslinker dimethylpimeli-
dimate (DMP) for coupling is based on the protocol described in Harlow and Lane
( Harlow et al., 1999 ).
Amounts indicated are for one tube of coupled beads
For one TAP purification prepare four tubes.
For one-step immunoprecipitation used for mass spectrometry prepare one tube.
Volume of Protein A beads indicated is for settled beads, material received from
Biorad is a 1:1 slurry.
1. Equilibrate 120 m LAffi-Prep Protein A beads into PBST (PBS + 0.1%Tween-20)
by washing the beads three to four times with 1 mL PBST. This should give about
60 m L of packed beads. Wash beads by gentle inversion. Briefly pellet them using a
pulse in a microcentrifuge (30 s at 3000g) and remove supernatant avoiding bead
pellet.
2. Resuspend beads in 500 m L PBST and add 10-50 m g of affinity-purified anti-
body. Mix for 45 min - 1 h at RT on a rotor to allow antibodies to bind to resin.
3. Wash beads three times with 1 mL PBST as described in step 1.
4. Wash beads three times with 1 mL 0.2 M sodium borate (pH 9.0) (dilute from a
stock of 1 M sodium borate (pH 9.0)). After the final wash, add 900 m L of the
0.2 M sodium borate (pH 9.0) to bring the final volume to 1 mL.
5. To initiate coupling add 100 m L of 220 mM DMP. Rotate tubes gently at RT for
30 min.
6. To make DMP: Let bottle sit tightly closed at RT for 20 min before opening.
Weigh out DMP and leave dry until just before use. Resuspend in appropriate
volume of 0.2 M sodium borate (pH 9.0) and add immediately to the bead
suspension (e.g., for 34 mg DMP add 596 m L sodium borate).
7. After incubation with DMP, wash beads two times with 1 mL 0.2 M ethanol-
amine, 0.2 M NaCl (pH 8.5) to inactivate the residual crosslinker. Resuspend in
1 mL of the same buffer and rotate for 1 h at RT. Resuspend beads in 500 m Lof
the same buffer. Leave the beads in 0.2 M ethanolamine, 0.2 M NaCl (pH 8.5) at
4 C until use. Beads are stable at 4 C for at least one month.
B. Immunoprecipitation
To prevent proteolysis it is important to keep the beads on ice and cool all buffers
and tubes before use. Using higher stringency conditions (300 mM KCl) reduces