Load 30µl of sample in each well of a 1.5mm thick gel.Boil for 5 minutes at 95☌ and spin down the beads at maximum speed in a microcentrifuge for 5 minutes at room temperature. After the final wash, remove the supernatant and add 20µl of 2X SDS sample buffer.Wash the beads 3 times with ice-cold cell lysis buffer. Collect the agarose beads by pulsing in a microcentrifuge tube (two minutes at 5,000 rpm, 4☌).Mix 100-500µl of cell extract with antibody-protein A, G, A/G agarose and rotate the samples at 4☌ for about two hours.Adjust the protein concentration of the supernatant to 1-2mg/ml with lysis buffer.Spin lysates at 14,000 rpm in a pre-cooled centrifuge for 10 minutes and keep the supernatant.Freeze and thaw the samples with dry ice for two more cycles or sonicate for 15 seconds to ensure the full release of the proteins from the cells.Discard the supernatant and immediately add 800µl of ice-cold lysis buffer to the cells and vortex, then incubate for 30 minutes on ice.Collect cells and centrifuge at 1200 rpm for 5 minutes at 4☌.Spin down the protein A, G, A/G beads for two minutes at 5,000 rpm and wash the antibody-beads three times with cell lysis buffer.Place the antibody-protein A, G, A/G agarose mix on a shaker and rotate at 4☌ for one hour.Adjust antibody concentration to 5-10µg/ml in PBS and transfer 500µl of diluted antibody to 5-10µl of agarose beads for each sample.Wash the beads three times with cell lysis buffer. Wash protein A, G, A/G agarose beads with cell lysis buffer by pulsing in a microcentrifuge tube (two minutes at 5,000 rpm).Preparation of antibody-protein A, G, A/G agarose beads: Use at 1XĢ.25g Tris base, 10.5g Glycine 1g SDS, 200ml Methanol. Use at 1Xĭissolve 144g of Glycine, 30g of Tris base and 10g SDS in 800ml of distilled H 2O. For example, one can perform immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phospho-specific antibody or an acetylation-specific antibody).Ģ5mM Tris-HCl pH 7.5, 150mM NaCl, 0.1% Triton X-100, 2mM EDTA, 1µg/ml leupeptin, 1µg/ml aprotinin, 1mM Na 3PO 4,1mM PMSF, 5mM NaF, 3mM Na 4P 2O 4ģ12.5mM Tris-HCl (pH 6.8) 10% SDS (w/v), 250mM DTT, 50% Glycerol, 0.05% Bromophenol Blue (w/v). Immunoprecipitation can also be used to “enrich” a protein population prior to Western Blotting. Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics.
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