Purifying IgG1 from Hybridoma Supernatant: Difference between revisions

From Tanaka Wiki
Jump to navigation Jump to search
No edit summary
 
(3 intermediate revisions by the same user not shown)
Line 1: Line 1:
<h3>Materials:</h3>
== Material ==
<li>Supernatant (100-200 ml)
* Supernatant (100-200 ml)
<li>Peristaltic pump
* Peristaltic pump
<li>1 ml HiTrap ProteinG column (binding capacity = 25 ml)
* 1 ml HiTrap ProteinG column (binding capacity = 25 ml)
<li>PBS  (100 ml)
* PBS  (100 ml)
<li>10x PBS
* 10x PBS
<li>.1 M glycine pH 2.7 (25 ml)
* .1 M glycine pH 2.7 (25 ml)
<li>1 M Tris pH 9.0  (10 ml)
* 1 M Tris pH 9.0  (10 ml)
<li>10 Eppendorf tubes
* 10 Eppendorf tubes
<li>20% ETOH/H2O  (25 ml)
* 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.

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

  1. 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
  2. Pump atleast 5 ml of ddH2O through column at 1 ml per minute
  3. Pump 10 mls PBS through column
  4. Pass supernatant over the column twice. (Save supernatant)
  5. While waiting, prepare 10 labeled tubes for collecting fraction, and put 100 µl of 1M Tris pH 9.0 inside
  6. Wash column with 15 ml PBS, then stop pump
  7. 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
  8. Wash column with 10 ml PBS
  9. Wash column into 10 ml 20% ETOH and store at 4 C.
  10. Quantitate protein with BioRAD reagent and dialyze into PBS.
Retrieved from "https://wiki.tanakalab.org/index.php?title=Purifying_IgG1_from_Hybridoma_Supernatant&oldid=812"