Immunoprecipitation via Biotinylation

Contents:

Prepare cells preparation, by washing 3 times in PBS (centrifuge at ª1200 rpm 4oC). Resuspend cells at 10^8/ ml in PBS.

Biotinylation Of Cell Surface Proteins

Solubilization of Cells

Preparation of protein G-Sepharose

Immunoprecipitation

NHSS-biotin : sulfo-N-hydroxysuccinimide ester derivative of biotin.

Nonidet P40 buffer : 1% NP40, 1 mM EDTA, 50mM Tris-HCl, 150mM NaCl pH 8.0

Protease inhibitors: 0.0174g of phenylmethyl sulphonyl flouride(PMSF) made up to 1 ml in Iso-propanol (added at 5ul/ml of cells).

Aprotinin 5ul/ml of cells

Iodoacetamide 0.0925g in 1 ml of PBS (added at 5ul/ml of cells).

Western Transfer

Visualisation of Biotin