We use PBS (sigma, pH 7.2-7.4) for this purpose.

Hi Nisha,

Here is my protein extraction protocol from cell pellet to perform an Elisa test: -Resumption of cell pellets in the extraction buffer RIPA (200uL around for a pellet of a BP100 box) -Let the pellets rest in the ice for 2 hours with stirring -Centrifugation 20 min, 16000g, 4 ° C -Recover the supernatant -Preserve the supernatants at -20 ° C RIPA buffer: - Tris 50mM, pH 8 - 150mM NaCl - IGEPAL CA-630 1% - Deoxychelate sodium 0.5% - SDS 0.1% + cocktail inhibitors

Thank you

If you have removed soluble proteins during lysis, such a pellet consists of membranes, cytoskeleton but also debries. The former two can be solubilised in a mild detergent, a further spin will remove the latter. The detergent may interfere with protein binding to plates, if you use standard plates that work by hydrophobic interaction. In general I found dot blot assays more convenient: You pass the protein solution over a PVDF membrane in a 96 well manifold, then develop the membrane like a western blot. Because the capture of protein is near quantitative (in my hands > 98%), sensitivity of dot blots is 2-3 orders of magnitude higher than ELISA. You need only one antibody against your protein, detergent doesn't interfere and you save a lot of incubation time.

As to the detergent to use, there are several considerations. Obviously, it needs to solubilise the protein you are interested in. Polyoxethylene detergents (Tween, Triton, CxEn) are mild, do not mobilise for example proteins in rafts, but may (?!) not interfere with capture antibodies. Anionic detergents like SDS are harsher. A compromise may be cationic detergents like quarternary ammonium (e.g. CTAB), amine oxides (LDAO, LAPAO) or pyridinium (CPC). They efficiently solubilise, but often leave enzymatic activity (a good marker of structure) intact.

It also depends on the antibodies you have. If they were raised against the native protein, they may not recognise proteins denatured by harsh detergents. If, on the other hand, they were developed against denatured protein (e.g., bands from SDS-PAGE), they may not recognise the native protein. The leaflet that came with the antibody should tell you that.