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| ==Isolation of Periplasmic Fraction==
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| Protocol courtesy of Yuri Londer, NEB
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| Make sure these chemicals are available:
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| #2xYT and antibiotics
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| #ice-cold TES
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| #protease inhibitor (NO REDUCING AGENTS)
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| #lysozyme
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| #ice-cold H<sub>2</sub>O
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| ===Cell Growth===
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| #In the AM, inoculate 5mL of LB with the glycerol stock of choice and appropriate antibiotics. Incubate at 37ºC and 250 rpm.
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| #In the afternoon, inoculate fresh 2xYT with appropriate antibiotics with 50uL of the first culture. Inoculate 10mL total. Grow at 30degC and 200rpm o/n.
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| #Prepare and autoclave flasks with 1 L of 2xYT media per flask.
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| #Next morning use 10 ml of the overnight culture to inoculate each 1L 2xYT with appropriate antibiotics. Let the cultures grow at 30ºC and 200 rpm for 24 hours. Do not induce.
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| #Next morning take out 2x 100 µl aliquot from each culture, centrifuge 5 min in a benchtop centrifuge, discard the supernatant and freeze the pellet.
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| #*This is for "whole cell" extracts in gels
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| #*If you'd like to compare this to B-PER, aliquot another 5mL of each culture.
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| #Harvest the bulk of the cells by centrifugation for 15 min at 4000 rpm, 4ºC; SAVE THE SUPERNATANT
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| #Resuspend the cells with a rubber policeman (see Note 35) in 30mL ice-cold TES buffer containing the amount of protease inhibitors recommended by the manufacturer
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| #*use 30 ml of TES buffer per pellet from 1 L of culture.
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| #*protease inhibitor:_______
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| #*Use the flattened tip of the rubber policeman to rub the cell pellet against the bottom (or walls) of the centrifuge bottle until there are no visible clumps of cells.
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| #*The efficiency of lysis depends on the efficiency of resuspending the pellet to near homogeneity.
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| #Transfer the suspension to 250 ml centrifuge bottles. Add lysozyme to a final concentration of 0.5 mg/ml (300 µl of a 50 mg/ml stock solution per bottle) and incubate for 15 min at room temperature
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| #*Alternatively, you can help solubilize the cytochromes with NaCl ___________
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| #*Avoid higher lysozyme concentrations and much longer incubation times as they may result in breakage of the inner membrane and contaminating the periplasmic fraction with the cytoplasmic content, including the chromosomal DNA.
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| #Add an equal volume (30 ml/bottle) of ice-cold water. Make sure that the lids are closed tightly and put the bottles horizontally in a bucket of ice. Shake gently (~ 100 rpm) for 15 min.
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| #Centrifuge at 12,000×g for 20 min at 4°C. Transfer the supernatants to fresh tubes or beakers.
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| #*. If a supernatant is viscous and difficult to transfer, add 5 µl of Benzonase Nuclease, incubate 15-30 min at room temperature and repeat the centrifugation step.
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| #Determine the spectra and A280 for each sample. Calculate the ratio of the Soret band absorbance over A280.
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| *'''[[User:Heather M. Jensen|Heather M. Jensen]] 09 Aug 2009 (CST)''':
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| Return to [[NanoBio:Protocols|Protocols]].
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