Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 315

1 mM MgCl 2
100 mM KCl
10% glycerol
0.05% NP-40
DTT goes off with time, so add it to the buffer just before the experiment.
Just prior to use add one tablet Complete EDTA-Free Protease Inhibitor
Cocktail Tablets (Roche Applied Science, #1873580) to 12 mL Lysis Buffer.
Use Lysis Buffer with 300 mM KCL as indicated in text.
3x Sample Buffer
6% SDS
240 mM Tris (pH 6.8)
30% Glycerol
0.04% (w/v) Bromophenol blue
Add 50 m L 100% 2-ME to 1 mL just before use.
1 M DTT
Dissolve 7.7 g DTT in 50 mL sterile ddH 2 O. Store 1 mL aliquots at -20 C.
Pre-urea Wash Buffer
50 mM Tris (pH 8.5)
1 mM EGTA
75 mM KCl
Filter to sterilize and store at RT.
Urea Elution Buffer
50 mM Tris (pH 8.5)
8 M urea (Invitrogen Cat. # 15505-035)
Store at RT and make fresh on the day of use.
2 M Tris (pH 8.5)
Dissolve 121.1 g Tris base in 800 mL ddH 2 O and adjust pH to 8.5 with HCl. Add
ddH 2 O to 1 L. Sterilize by autoclaving and store at RT.
100% TCA (Sigma Aldrich Cat. # T0699100 mL)
E. Tandem Affinity Purification using a LAP Tag
GFP antibody
TEV protease 6His-TEV protease (1 mg/mL stock), purified using nickel-nitrilo-
triacetic acid (Ni-NTA) agarose and gel filtration ( Kapust et al., 2001; Parks et al.,
1995 ), or available from Invitrogen (Cat. #12575-015)
S protein agarose (Novagen Cat. #69704)