| Antibody Production and Denaturing Purification of proteins not soluble in PBS James Fishburn, Hahn Lab March 2006 Proteins should be submitted at 1.0 mg/ml (4 mg total) in PBS A small quantity of purified denatured protein should be kept for testing the generated antibodies If proteins are not soluble in PBS they can be purified under denaturing conditions, dialyzed in PBS, sonicated, and submitted as precipitates. Apparently, an emulsification process that the proteins go through before injection allows this to work. Proteins with a 6-His N-terminal tag are expressed from pET21(a) in BL21(DE3)RIL cells Denaturing Buffers Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 6 M Guanidinium-HCl, 20 mM imidazole pH 7.4 Wash buffer: 20 mM sodium phosphate, 0.5 M NaCl, 8 M urea, 20 mM imidazole pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 8 M urea, 500 mM imidazole pH 7.4 Day 1 Grow 10+ ml culture overnight Day 2 1. Inoculate 1 L media with 10 ml saturated overnight 2. Grow cells to OD600 0.6 (ca. 3hrs at 37 deg) 3. Induce cells with 1 mM IPTG 4. Continue incubation for 3 hrs at 30 deg with shaking 5. Harvest cells- 5' spin at 5000 rpm in GSA 6. Resuspend cell pellet in 30 ml Binding buffer + 10 mM beta-mercaptoethanol 7. Incubate extract at RT with gentle mixing until translucent 8. Clarify extract by centrifugation: 20' spin at 10,000 rpm in SS-34 9. Transfer supernatant to 50 ml tubes, freeze in N2, and store at -70 deg Day 3 1. Thaw samples in RT water bath 2. Make Wash and Elution buffers during thaw 3. Prepare Ni-Sepharose: wash with 5 volumes DI water, wash with 5 volumes Binding buffer, resuspend Ni-Sepharose to 50% in Binding buffer (spins are at 500 x g for 2'-5') 4. Add 2 ml 50% Ni-Sepharose slurry to extract 5. Incubate extract with resin for 30' at RT with mixing (nutate) 6. Collect resin by centrifugation, remove supernatant and save (flow through) 7. Wash resin a total of three times using 5 volumes of Binding buffer for each wash 8. Add 2 volumes Elution buffer to resin and incubate 5' at RT with mixing (nutate) 9. Collect resin and save first elution 10. Repeat elution step 2-4 more times saving each elution 11. Analyze purification by SDS-PAGE (4-12% Bis-Tris, MES) and CBB staining- 2 ºl of each elution per lane is sufficient for gel staining along with 1 ºl each of crude lysate, clarified lysate, and the flow through 12. Combine desired elutions 13. Determine concentration of protein by Bradford assay 14. Dilute protein to 1 mg/ml in PBS + 3 M urea 15. Dialyze 5 ml of protein in 500 ml PBS + 1 mM PMSF + 1 mM DTT for 1 hr at RT 16. Repeat dialysis two more times (3 x 1hr total): protein will precipitate 17. Transfer dialyzed and precipitated protein to 12 ml Falcon tube (#352059) 18. Put protein on ice 19. Sonicate protein using small tip for 2 x 20 second pulses with 1 minute incubation on ice between pulses 20. Divide protein into 5 x 1 ml aliquots in 1.5 ml tubes 21. Freeze and store at -70 deg |
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| Last Updated 4/18/06 | ||||||
