Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 313

1 M potassium citrate (pH 6.0)
268.8 g tripotassium citrate
26.3 g citric acid monohydrate
Add ddH 2 O to 900 mL, adjust pH to 6.0 using 10 N KOH and add ddH 2 Oto
1 L. Autoclave and store at RT.
1 M MgSO 4
1 M CaCl 2
Complete S Basal
Working under sterile conditions in the laminar flow hood add the following to
500 mL S Basal:
5 mL 1 M potassium citrate (pH 6.0)
5 mL Trace Metals Solution
1.5 mL 1 M MgSO 4
1.5 mL 1 M CaCl 2
C. Isolation of Nuclei from Embryos
30% (w/v) sucrose in Nuclei Buffer
Embryo Buffer
25 mM Hepes KOH (pH 7.6)
118 mM NaCl
48 mM KCl
2 mM CaCl 2
2 mM MgCl 2
Nuclei Buffer
10 mM Tris:HCl (pH 8.0)
80 mM KCl
2 mM EDTA
0.75 mM spermidine
0.3 mM spermine
Spermine and spermidine are prepared as 1 M stocks in ddH 2 O and stored at -
20 C. Add immediately before use.
Add 1 tablet Complete EDTA-Free Protease Inhibitor Cocktail Tablets (Roche
Applied Science, Cat. #1873580) per 10 mL buffer just prior to use.
10% (w/v) digitonin
(Sigma, Cat. #37006) stock solution is prepared in ddH 2 O by boiling and
filtering. Store aliquots at - 20 C. Briefly boil, chill on ice, and add to
Nuclei Buffer immediately before use.