Aug 05, 2020
Protein Extraction
- 1domnic colvin
- UM-DAE Centre for Excellence in Basic Sciences
Document Citation: avinash.kale 2020. Protein Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bjdnki5e
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 05, 2020
Last Modified: August 05, 2020
Document Integer ID: 40078
Protein Extraction
The four Bacillus bacteria were inoculated in 200 ml LB medium (Serva) and incubated at 37ºC overnight. The cells were centrifuged at 13000 rpm for 15 minutes at 4°C to pellet the cells. The cell pellet was sonicated at amplitude 30%, pulse 10 seconds, gap 10 seconds, for a total time of 2 minutes: 30 seconds or 15 Cycles to obtain the cell lysate. The TRIzol extraction protocol was used with minor modifications(24). In brief, to the cell lysate, 1 mL of TRIzol reagent and 200 µL of chloroform were added. The solution was mixed vigorously and was centrifuged 14,000g for 15 minutes at 4°C. The Upper clear phase was carefully decanted and to the remaining phase, 300 µL of ethanol was added and incubated at room temperature for 3 minutes followed by centrifugation at 2000g for 15 minutes at 4 °C. To the supernatant, a 4-fold volume of ice-cold acetone was added and kept for overnight incubation at -20ºC. The protein pellet was washed thrice first with 0.3 M Guanidium-HCl in 95 % ethanol followed by ice-cold acetone two times. All the protein pellets were air dried and wer subsequently dissolved in rehydration buffer (7 M urea, 2 M thiourea, 2 % CHAPS, Distilled water). Protein concentrations were estimated using Bradfor assay.