Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 314

Chitinase
Dilute in Embryo Buffer at 2 U/mL and store aliquots at - 20 C. The efficiency
of chitinase treatment varies and the optimal length of digestion should be re-
determined for each new batch.
Hoechst 33342
D. Single-step Immunoprecipitation
Antibody coupling
10-50 m g of affinity-purified antibody
Affi-Prep Protein A beads (Biorad Cat. #156-0006)
PBST (PBS + 0.1% Tween-20)
1 M sodium borate (pH 9.0)
Dissolve 61.8 g of boric acid in 800 mL of ddH 2 O and adjust the pH to 9.0 with
NaOH pellets. Bring volume to 1 L with ddH 2 O and filter to sterilize. Store
at RT.
0.2 M sodium borate (pH 9.0)
Dilute from a stock of 1 M sodium borate (pH 9.0).
220 mM DMP
(Sigma Cat. #D8388; FW259.2; stored in a dessicated box at 20 C) 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).
0.2 M ethanolamine, 0.2 M NaCl (pH 8.5)
Dissolve 12.2 g of ethanolamine and 11.7 g of NaCl in ddH 2 O, adjust the pH to
8.5 with HCl. Add ddH 2 O to 1 L and filter to sterilize. Store at RT.
Immunoprecipitation
100 mM glycine (pH 2.6)
Dissolved 7.5 g glycine in 800 mL of ddH 2 O and adjust pH to 2.6 with HCl. Add
ddH 2 O to 1 L. Filter to sterilize and store at 4 C.
Lysis Buffer
50 mM HEPES (pH 7.4)
1 mM EGTA