Purifying IgG1 from Hybridoma Supernatant: Difference between revisions
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== Material == | |||
* 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) | |||
== 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 | ||
Latest revision as of 08:48, 19 June 2015
Material
- 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)
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.