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viral immunity and pathogenesis group. antibody purification with protein G.


l a b o r a t o r y   r e s e a r c h   i n   i m m u n o l o g y   ::   v i r o l o g y   ::   h e m a t o l o g y
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v i r a l   i m m u n i t y   &   p a t h o g e n e s i s   g r o u p

::  p r o t o c o l s  ::  Ab  p u r i f i c a t i o n   ::







[  ammonium  sulphate  precipitation  ]

OUTLINE

Ammonium sulphate precipitation is used to purify a protein (in this case an immunoglobulin) from a big volume of  liquid phase.

PROTOCOL

  1. Slowly (!!!) add the solution of ammonium sulphate (40-50% final, v/v) to hybridoma cell culture supernatant (60-50% final, v/v). Store at 4C ,ON (overnight).
  2. centrifuge the precipitant at 5000-10000 rpm for 10-15 min at RT.
  3. resuspend the precipitant in PBS (as small volume as best).
  4. dialyse against PBS, ON for 48 hours.
  5. filter on 0.22 m filter.
  6. run ELISA to establish the Ig concentration

SOLUTIONS

  1. ammonium sulphate, saturated, pH 6.8, store at RT
  2. PBS


[  p u r i f i c a t i o n  o n  p r o t e i n  G  ]

OUTLINE

Protein A (from Staph. aureus) and protein G (from Streptococcus sp. [Lancefield Group G]), both exhibit an affinity for the Fc portion of  diverse array immunoglobulins from many species. Protein A binds to Fc-gamma and Fv-VHIII . Protein G binds to Fc-gamma and c-gamma1 chains.

PROTOCOL

Step Description Details
Step 0 - Delipidtion
  1. Use this step when purifying ascites or serum
  2. Use SeroClear : ascitesFluid/serum =1:1,5->vortex 1min,RT->spin 5000rpm,10min-> retain the top layer for subsequent purification
  1. SeroClear- CalBiochem cat. No.437616, not compatible with polysterene tubes
  2. Alternatively mix 3:2 = 1,1,2-trichlorotrifluoro -ethane:serum/ascitesFluid->shake 30min,RT,spin 5000rpm,RT,10min -> retain the top layer for subsequent purification
  3. Alternatively for ascitesFluid use a glass wool by placing into a funnel to cover the opening, pouring ascites through, rinsing glass wool with PBS, and squeezing glass wool gently with gloved fingers to obtain all the sample. Centrifuge filtered ascites 30min at 20000g ,RT or 4C. Decant and save the SN.
Step 1 -
Equilibration
& Loading
  1. Equilibrate the column with 5-10 CV(column volums) of 20mM sodium phosphate , pH7
  2. Dilute ascites or serum 1:1 with 20mM sodium phosphate , pH7, !FILTER 0,8->0,45-0,22um and then load onto the column
  3. Loading rate : 50ml/hr (0,8ml/min)
  1. Alternatively it`s possible to use PBS x 1 instead of 20mM sodium phosphate or 0.1M sodium acetate , pH5.0 or 10mM sodium phosphate, pH 7.0 with 0.15M NaCl for equilibration-loading-washing
  2. alternatively it`s possible to incubate the gel directly with serum or ascitesFluid for 30min before applying the gel to the column
  3. alternatively dilute 2-fold for tissue culture SN or 10-fold for ascitesFluid and bioreactor SN in the loading buffer
Step 2 - Whashing
  1. wash the column with 20mM sodium phosphate , pH7
  2. collect the flow-through
  1. usually 5-10CV are used to wash the column
  2. check the OD280 of the flow through to determine when whashing is complete
Step 3 - Elution
  1. elute the bound Abs with 0.1M glycine-HCl ,      pH 2.7 (2.2)
  2. collect fractions and monitor the OD280
  3. collect fractions into tubes containing 50-100ul 2M TRIS base , pH8.0per 1ml of fraction , pool fractions
  1. alternatively use 0,1M acetic acid , pH 2.8 to elute
  2. Ab can be stored in 2M TRIS base , pH8.0,or can be exchanged for PBS x 1 or TRIS buffer by dialysis
  3. Add 0,05% NaN3 if stored at 2-8C and 50% glycerol for -20C
Step 4 -Washing, Cleaning and Gel storage , Re-equilibration
  1. Wash with 0.1M glycine-HCl , pH 2.7
  2. Clean with C2H5OH 70%-wash , incubate for 12hrs
  3. Store with 20% C2H5OH, store at 2-8C, don`t freeze    
  4. Re-equilibration with 20mM sodium phosphate , pH7
  1. alternatively use 1M acetic acid for washing (3-4CV)
  2. alternatively use 100mM PBS , pH7.4 with Proclin 0.01% as a preservative for storage
  3. alternatively use 0.1M sodium acetate , pH5.0 for re-equilibration


SOLUTIONS

Buffer

Components & details

20mM sodium phosphate, pH 7.0

3,27g Na2HPO4 x 7H2O

0,94g NaH2PO4

q.s. ddH2O to 1L

correct pH to 7.0

! Use NaOH or HCl to adjust pH being careful not to overshoot and back-titrate, as this may alter salt concentration more than necessary

10mM sodium phosphate, pH 7.0 with 0.15M NaCl

1.64g NaH2PO4 x 7H2O

0.47g NaH2PO4

8.77g NaCl

q.s. ddH2O to 1L

correct pH to 7.0