Embryo Lysates & Immunoprecipitation

ough you will generally use more for IPs and DNA fishing.MKs modifiedlysis buffer 50 mM Tris pH 8.0150mM NaCl0.5% NP400.5% Triton-X1001mM EGTA5mM NaF + protease inhibitorstop of pageImmunoprecipitation with protein A agaroseUse lysates prepared using freon extraction add 0.5-1 µL of antibody - incubate with end-over-end rotation for 1-4 hours at 4°C. add 40µL protein-A-agarose, incubate with end-over-end rotation for 1-2 hours at 4°C. recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm. Remove lysate by aspiration --it is ok to leave some liquid above the pellet. resuspend in your original lysis buffer (1.5ml) and wash for 5 min. at 4°C (with end-over-end rotation) recover beads by centrifugation, at 4°C, 2 min. at 2000 rpm resuspend in high salt wash buffer (buffer 2) and wash for 5 min. at 4°C recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm High salt buffer: 50 mM Tris-HCl pH 7.5 500mM NaCl 0.1% Nonidet 40 0.05% sodium deoxycholate Low salt buffer: 50 mM Tris-HCl pH 7.5 0.1% Nonidet 40 0.05% sodium deoxycholate resuspend in low salt wash buffer (buffer 2) and wash for 5 minutes at 4°C recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm remove supernatant as completely as possible without disturbing beads. spin again - remove supernatant add 1X SDS sample buffer + ßME - heat at 80°C for 5 minutes - spin beads to bottom of tube load on gel! top of pageImmunoprecipitation with protein A magnetic beads for silver stain/sequencing analysisprepare lysates (using freon extraction) absorb lysate by running it over a µMacs column (You can omit this step if you are not going to attempt silver staining or sequence analysis). add 0.5 µL of antibody - incubate with end-over-end rotation for 1-4 h at 4°C. add 50-100µL protein-A-magnetic beads (Miltenyi), incubate with end-over-end rotation for 1h to overnight at at 4°C. reequibrate fresh columns with lysis buffer (all subsequent steps are done at room temperature). run supernatant + antibody + beads over column. wash 4 x 200 µL with lysis buffer add 20 µL Hot 1X SDS-sample buffer - let sit for 5 minutes. add 50-80 µL hot 1x SDS-sample buffer and recover flow through. This is your sample! load sample on gel!
< 1 > < 2 >

设为首页 | 加入收藏 | 广告服务 | 友情链接 | 版权申明