Team:USTC CHINA/Notebook/Protocols/Sample analysis

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<h1>Sample analysis</h1>
<h1>Sample analysis</h1>
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<p>Sample analysis
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<p>
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1. Preparation of soluble and insoluble cell extracts from B. subtilis
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<span>1. Preparation of soluble and insoluble cell extracts from B. subtilis</span></br>
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- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)
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- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)</br>
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- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10
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- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10</br>
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- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml
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- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml</br>
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Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme
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Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme</br>
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(250 μg/μl, CB-0663-5GAM), on ice
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(250 μg/μl, CB-0663-5GAM), on ice.</br>
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破碎1:超声 
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- alternatively, cells can be disrupted by beat beating:
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- alternatively, cells can be disrupted by beat beating:</br>
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disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in
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disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in</br>
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an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption
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an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption</br>
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破碎2:玻璃珠机械破碎
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- take 100 μl of the preparation as first total protein sample (T1)
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- take 100 μl of the preparation as first total protein sample (T1)</br>
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得到T1
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- remove cell debris by centrifugation at 4,300 x g, 10 min
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- remove cell debris by centrifugation at 4,300 x g, 10 min</br>
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- take 100 μl of the supernatant for the second total protein sample (T2)
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- take 100 μl of the supernatant for the second total protein sample (T2)</br>
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- spin at 8.200 x g (10 min, 4 °C)  
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- spin at 8.200 x g (10 min, 4 °C) </br>
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to separate into insoluble (I) and soluble (S) protein fractions.
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to separate into insoluble (I) and soluble (S) protein fractions.</br>
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- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by
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- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by SDS-PAGE</br>
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SDS-PAGE
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- analyze samples by immunoblotting with specific antiserum</br>
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- analyze samples by immunoblotting with specific antiserum
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Latest revision as of 09:12, 27 September 2013

Sample analysis

1. Preparation of soluble and insoluble cell extracts from B. subtilis
- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)
- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10
- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml
Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme
(250 μg/μl, CB-0663-5GAM), on ice.
- alternatively, cells can be disrupted by beat beating:
disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in
an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption
- take 100 μl of the preparation as first total protein sample (T1)
- remove cell debris by centrifugation at 4,300 x g, 10 min
- take 100 μl of the supernatant for the second total protein sample (T2)
- spin at 8.200 x g (10 min, 4 °C)
to separate into insoluble (I) and soluble (S) protein fractions.
- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by SDS-PAGE
- analyze samples by immunoblotting with specific antiserum