Immunopurification and Immunoprecipitation
1. Plan conditions for dissociation of sample. Some standard conditions are as follows:
Buffer Detergents DTT Dissociation Conditions
Mild IP buffer 1% TX-100 (0.5 mM) 4 - 25°C for 5-60 min.
Standard IP buffer 1% TX-100, 0.2% SDS (1 mM) 4 - 50°C for 5-60 min.
Harsh IP buffer 1% SDS 5 mM 37 - 100°C for 2-5 min.
IP buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 8. Protease inhibitors should be freshly added.
IP buffer can also include 1 mM EDTA (to dissociate proteins from RNA) or MgCl2 (to stabilize
NP-40 can be used in place of TX-100.
DTT in "mild" and "standard" is optional (0.5-1 mM is okay for most antibodies). Other
conditions may be better for your application. Conditions for IPs must be empirically determined
in each case. The conditions above are intended as initial ones to be tried first. Other conditions to
consider are: urea at concentrations below 0.5 M; NaCl or KCl at concentrations up to 1 M; pH of
6 to 9; addition of 0.5% sodium deoxycholate (for membrane-bound antigens); addition of 5 mM
iodoacetamide instead of DTT (this sulfhydryl blocker may inactivate some enzymatic activities);
addition of BSA or other blocking agents.
2. Prepare antibody-sepharose conjugate ahead of time. This may be done by:
A. Binding antibody to protein A-sepharose or protein G-sepharose.