IMMUNOPRECIPITATION |
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Last Update: December 2006 |
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| PROCEDURE | |
| 1. Samples (500 mL) are solubilized by addition of 20 mL 10% SDS (to final 0.4%), 5 mL of 0.1mM PMSF, and 50 mL (20% Triton X100, 1.5M NaCl, 0.3 M Tris-HCl, pH 7.5). Mix well (brief vortex), place on a rocker and incubate at 25oC for 10-15 minutes | |
| 2. Spin cell debris for 2 minutes in a micro-centrifuge at 1000 xg. Collect the supernatant into new tube | |
| 3. Add 20 mL of anti-sera [as control use pre-immune sera], place on a rocker and incubate for 2 hours at room temperature (or over-night at 4oC) | |
4. Add 100 mL of a 50 % (v/v) suspension of protein-A sepharose beads previously equilibrated with 20mM Tris-HCl, pH 7.5 , 0.15M NaCl. 0.5% Triton X-100 |
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| 5. Spin for 15 seconds at full speed, discard the immuno-precipitate FT supernatant | |
| 6. Wash the beads 3 times (for 2-10 mins) with 1 mL of 20mM Tris-HCl pH 7.5, 20mM EDTA, 0.5 % Triton X100, 0.25% SDS, 150mM NaCl). After the last wash remove as much supernatant as possible | |
| 7. Elute the proteins from beads by adding 30 mL of 125mM Tris-HCl, pH 6.8, 2% SDS, 10% Glycerol, 5% b-mercaptoethanol, and 0.01% bromophenol blue. If the eluted protein is not a membrane bound incubate for 2 min at 95oC. If it is a membrane protein incubate for 5 mins at 37oC | |
| 8. Spin for 10 seconds, collect the eluted protein and analyze sample by SDS-PAGE (SDS/Native PAGE (112)) | |