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dandan_hui木虫 (正式写手)
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[交流]
【求助/交流】重金求关于PULL-DOWN 的protocol一篇
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| 求各位同仁给一篇pull-down 的protocol，在网上找了好久也没找到，谢谢大家啦 |
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snowice
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scelab(金币+3):谢谢交流！:tiger06: 2010-09-02 23:00:29
dandan_hui(金币+1): 2010-09-03 08:14:24
scelab(金币+3):谢谢交流！:tiger06: 2010-09-02 23:00:29
dandan_hui(金币+1): 2010-09-03 08:14:24
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GST-Fusion Protein Purification and Pull Down Assay 1. Grow BL21 bacteria containing plasmid(from a single colony or -70°C stock,) in 5ml LB plus 50ug/ml ampicillin at 37°C shaker for overnight; 2. Inoculate 400ml of LB plus 50ug/ml ampicillin with 4mlo/n culture; 3. Grow for 2-3 hrs until OD600 is 0.5; 4. Add IPTG to a final concentration of 0.1 and grow at 37°C for 2-3hrs(update, except inducing GST-Rhoteckin at 30°C for 3hrs because of its unstability); 5. Spin 6k rpm for 15 min 4°C and resuspend the pellet in 10ml of PBS plus 1mM PMSF and 10mM benzamidine; 6. Sonicate 5x30sec, at maximum power; Incubate the sonicates in ice between sonications for 30sec; 7. Add Triton X-100 to 1% and DTT to 1mM (both final concentration) and incubate for 30min in ice; 8. Spin 7k rpm for 10min at 4°C. Take the supernatant for the next step and save the pellet for inclusion body purification. 9. Transfer the supernatant into a 50 ml conical tube, add 25ml in PBS plus 1mM PMSF and 10mM Benzamidine and 2ml 1:1 slurry of glutathione-agarose beads. (If make GST fusion protein from 5ml culture, reduce the PBS volume to 1ml and add 50ul glutathione-agarose beads) The beads need to be preswollen overnight in PBS and washed 4 times with PBS before use. 10. Incubate at 4°C on a rotator for overnight; 11. Wash the beads with PBS at 4°C for 5 times. 12. Batch elute with 1 column volume of 20mM glutathione in 20mM Tris, pH8.0 for 6 times. 13. Pool and dialyze against 4 liters of PBS o/n at 4°C. 14. The GST fusion protein can be estimated by measuring the absorbance at 280nm. For the GST affinity tag, 1A280=0.5mg/ml. Also, it can be determined by SDS-PAGE with standard BSA protein. 15. Incubate about 500mg cell lysate with 2-5ug GST fusion protein and 40ul Gluthatione-agarose beads(if the GST fusion protein-binding beads are less than 40ul, use empty beads to 40ul) in a total 1ml volume(adjust to 1ml volume with RIPA buffer ) at 4°C on a rotator for at least 1hr. Briefly wash the beads with Modified RIPA buffer for 3 times. 16. Elute the protein into the SDS loading buffer, then run SDS-PAGE gel. Reagent: 1. Glutathione-Agrose beads: G-4510 from Sigma. 2. Glutathione, reduced form: G-6529 from Sigma. 3. Benzamidine: B-6506 from Sigma, 1M stock solution. 4. PMSF(phenylmethyl-sulfonyl fluoride): P7626 from Sigma, 0.1M stock solution in isopropanol. 5. RIPA buffer:150mM NaCl, 50mM Tris.HCl(PH7.5), 1%NP-40, 0.25% Sodium Deoxyclolate, 2mM EGTA(stock solution 0.4M add before use), 1mM NaVO4(200mM stock solution, add before use) 6. 6XSDS loading buffer: 0.35M Tris-HCl(pH6.8), 10.28(w/v)%SDS, 36%(v/v) glycerol, 0.6M dithiothreitol(or 5%β-mercaptoethanol), 0.012%(w/v)bromophenol blue. Store in 0.5ml aliquots at -80°C. |
2楼2010-09-02 22:46:17
biotech113
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dandan_hui(金币+9): 2010-09-03 08:14:20
lstt09nk(金币+1):很热心~~ 2010-09-03 09:52:22
dandan_hui(金币+9): 2010-09-03 08:14:20
lstt09nk(金币+1):很热心~~ 2010-09-03 09:52:22
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我这儿有个GST-Pull-Down 的Protocol, 你可以参看下 http://d.namipan.com/d/b8f32bd3e ... bcaa9b580d1b6780400 |
3楼2010-09-02 23:03:53
