OVERVIEW
Co-immunoprecipitation requires intact interaction among proteins in order to co-immunoprecipitate protein complexes with the target protein. To keep the interactions intact, a special cell lysis buffer is required which can disturb the protein-lipid interactions but retains the protein complexes in their native form without causing denaturation of proteins. One of the common buffers used for Co-IP contains 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol.
REQUIREMENTS
Reagents and solutions
1M Tris-Cl, pH 7.4
5M Sodium chloride (NaCl)
0.5M EDTA, pH 8.0
10% NP-40
Glycerol (99.9%)
Double distilled water/MilliQ water
Equipment and disposables
Falcon® screw cap graduated Conical Tubes, 50 mL (e.g., SARSTEDT #62.547.254)
Pipette tips and micropipette
COMPOSITION
25 mM Tris-HCl pH 7.4
150 mM NaCl
1 mM EDTA
1% NP-40
5% glycerol
OBJECTIVE
Preparation of 50 ml of Co-IP lysis buffer
PREPARATION:
Use personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow the guidelines of your institute.
Step 1: Take some water (≈ 25 ml) in a 50 ml Falcon® screw cap graduated Conical Tubes and add 1.25 ml of 1M Tris-Cl (pH 7.4), 1.5 ml of 5M Sodium chloride (NaCl), 0.1 ml of 0.5M EDTA (pH 8.0), 5 ml of 10% NP-40 and 5 ml of 99.9% glycerol. Mix it.
Note: Don’t mix concentrated solutions together that can cause the precipitation of salts. It is recommended to add them in a sufficient amount of water (≈25 ml) as we have described in step 1.
Step 2: Adjusted the volume to 50 ml with MilliQ water.
Note: Since we have used 50 ml Falcon® screw cap graduated Conical Tubes, you do not need to transfer the solution to a measuring cylinder.
STORAGE
Store the solution at 4°C. Solution is stable for a year.
