Protein Expression and Purification Series - Bio-Rad
Protein Expression and Purification Series - Bio-Rad
Protein Expression and Purification Series - Bio-Rad
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<strong>Protein</strong> <strong>Expression</strong> <strong>and</strong> <strong>Purification</strong> <strong>Series</strong><br />
Lab<br />
Session<br />
Timeline for the Laboratory Course:<br />
Centrifugation <strong>Purification</strong><br />
Centrifugation <strong>Purification</strong> continued<br />
Chapter Task Estimated<br />
Duration<br />
5 4: Culturing,<br />
<strong>Expression</strong>, Lysis <strong>and</strong><br />
SDS-PAGE Analysis<br />
for 11 ml Cultures <strong>and</strong><br />
<strong>Purification</strong> Protocol for<br />
Centrifugation Process<br />
6 4: Culturing,<br />
<strong>Expression</strong>, Lysis <strong>and</strong><br />
SDS-PAGE Analysis<br />
for 11 ml Cultures <strong>and</strong><br />
<strong>Purification</strong> Protocol for<br />
Centrifugation Process<br />
Place resuspended cells at<br />
–20°C<br />
21<br />
16+ hr* N/A<br />
Thaw completely 30 min N/A<br />
Place resuspended cells at<br />
–20° C<br />
16+ hr* N/A<br />
Thaw lysate completely 30 min N/A<br />
Separate soluble vs insoluble<br />
fractions via centrifugation<br />
20 min N/A<br />
Module Containing<br />
Materials<br />
Decant soluble fraction 10 min Growth <strong>and</strong><br />
<strong>Expression</strong><br />
Resuspend insoluble fraction<br />
in lysis buffer<br />
Prepare soluble <strong>and</strong> insoluble<br />
SDS-PAGE samples<br />
Optional stopping point: Store<br />
fractions at –20°C indefinitely<br />
Prepare Micro-<strong>Bio</strong>Spin column<br />
with Profinity IMAC Ni-charged<br />
resin<br />
Bind GST-DHFR-His soluble<br />
fraction to IMAC Ni-charged<br />
resin<br />
Collect flowthrough, wash, <strong>and</strong><br />
eluate fractions<br />
Prepare flowthrough, wash<br />
<strong>and</strong> eluate SDS-PAGE<br />
samples<br />
15 min Growth <strong>and</strong><br />
<strong>Expression</strong><br />
15 min SDS-PAGE<br />
Electrophoresis<br />
15–30 min Growth <strong>and</strong><br />
<strong>Expression</strong> <strong>and</strong><br />
Centrifugation<br />
<strong>Purification</strong><br />
20–30 min N/A<br />
15–30 min Growth <strong>and</strong><br />
<strong>Expression</strong> <strong>and</strong><br />
Centrifugation<br />
<strong>Purification</strong><br />
15 min SDS-PAGE<br />
Electrophoresis<br />
*Time indicated for these tasks is not h<strong>and</strong>s-on time. It is the time needed for reactions to run, bacteria to grow, or time to incubate.
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