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In-gel_digestion_procedure

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					In-Gel Digestion Procedure
1. Slice gel spots into cubes, transfer the gel particles into a 1.5 ml microcentrifuge tube, add 30 ul of H2O and leave at room temperature for 15 minutes. 2. Remove the supernatant, add 20 ul of 50% acetonitrile and leave for 15 minutes. 3. Remove the supernatant, add 30 ul of 100% acetonitrile and leave for 15 minutes. 4. Remove the supernatant, add 20 ul of 0.1 M NH 4HCO3 and leave at room temperature for 5 minutes. 5. Add 30 ul of 100% acetonitrile and mix for 15 minutes. 6. Remove the supernatant and completely dry the gel pieces with a Speed Vac (20 minutes) 7. Add 50 ul of 10mM DTT in 0.1 M NH 4 HCO3 and incubate at 56 0C for 45 minutes. 8. Remove the supernatant, add 30 ul of 55mM IAA(idoacetamide) in 0.1 M NH4HCO3 and leave in the dark for 30 minutes 4 0C. 9. (Repeat steps 2-6) 10. Add 20 ul of 0.1% RapiGestTM SF solution in 50mM NH 4HCO3 and incubate at 37 0C for 10 minutes. 11. Remove excess solution and completely dry the gel pieces with a Speed Vac (20 minutes) 12. Add Trypsin* (20ng/ul) to the gel slice until the gel is reswollen and incubate 45 minutes 4 0C 13. Remove excessive solution, add 20 ul of 50mM NH 4HCO 3 and incubate at 37 0C 16 hr 14. 3 times, 50 ul 0.1% TFA/60%ACN extraction(First time sonication 5 minutes) 15. Completely dry the digestion solution with a Speed Vac (1 hr)

*

Trypsin per vial contain 20 ug (SIGMA-Trypsin, Proteomics Grad, Product Code T 6567) add 900ul 50mM NH 4HCO3 +100ul acetonitrile become 20ng/ul.


				
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