Purifying IgG1 from Hybridoma Supernatant: Difference between revisions
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Supernatant (100-200 ml)
Peristaltic pump
1 ml HiTrap ProteinG column (binding capacity = 25 ml)
PBS (100 ml)
10x PBS
.1 M glycine pH 2.7 (25 ml)
1 M Tris pH 9.0 (10 ml)
10 Eppendorf tubes
20% ETOH/H2O (25 ml)
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== Materials == | |||
<li>Supernatant (100-200 ml) | <li>Supernatant (100-200 ml) | ||
<li>Peristaltic pump | <li>Peristaltic pump | ||
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<li>20% ETOH/H2O (25 ml) | <li>20% ETOH/H2O (25 ml) | ||
== Procedure == | |||
# Add 0.1 volume 10X PBS to supernatant and filter througn 0.22 µm filter to prevent clogging. Keep until ready to run on column | # Add 0.1 volume 10X PBS to supernatant and filter througn 0.22 µm filter to prevent clogging. Keep until ready to run on column | ||
# Pump atleast 5 ml of ddH2O through column at 1 ml per minute | # Pump atleast 5 ml of ddH2O through column at 1 ml per minute | ||
Revision as of 10:30, 27 March 2015
Materials
Procedure
- Add 0.1 volume 10X PBS to supernatant and filter througn 0.22 µm filter to prevent clogging. Keep until ready to run on column
- Pump atleast 5 ml of ddH2O through column at 1 ml per minute
- Pump 10 mls PBS through column
- Pass supernatant over the column twice. (Save supernatant)
- While waiting, prepare 10 labeled tubes for collecting fraction, and put 100 µl of 1M Tris pH 9.0 inside
- Wash column with 15 ml PBS, then stop pump
- Collect 1 ml fractions into the preprepared tubes by eluting in 0.1 M glycine, pH 2.7. Collect 10 fractions, and mix immediately with Tris to bring pH to 7.0
- Wash column with 10 ml PBS
- Wash column into 10 ml 20% ETOH and store at 4 C.
- Quantitate protein with BioRAD reagent and dialyze into PBS.