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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

0.1M glycine-HCl , pH 2.7

11.1g Glycine-HCl

800 ml ddH2O

q.s. H2O to1L

correct pH to 2.7

0,1M acetic acid , pH 2.8

5.27 ml Glacial Acetic Acid

800ml ddH2O

q.s. H2O to1L

correct pH to 2.8

2M TRIS base , pH8.0

 

PBS x 5 (dilute 5 times for PBS x 1) pH7.2

 

100mM PBS , pH7.4 with Proclin 0,01%

2.6g KH2PO4

21.7g Na2HPO4 x 7H2O

8.77g NaCl

1ml proclin

800ml ddH2O

q.s. H2O to1L

correct pH to 2.8


ADDITIONAL INFO
                                                                                                                                  
Filter all solutions on 0.8->0.45->0.22 m filter !!!

Binding properties of protein G and A

Ab sourse

Protein G

Protein A

Mouse mIgG1

++ (high)

+ (low)

Mouse mIgG2a

++ (high)

++ (moderate)

Mouse mIgG2b

++ (high)

++ (moderate-high)

Mouse mIgG3

++ (high)

+ (high?)

Mouse mIgM

?

+

Mouse mIgA

+

++

Mouse poIg

++

++

 

 

 

Rat mIgG1

(moderate)

(low)

Rat mIgG2a

(high)

(low)

Rat mIgG2b

(low)

(low)

Rat mIgG2c

(high)

(high)

Rat poIg

++

+

 

 

 

Rabbit poIgG

+++ (high)

++ (high)

 

 

 

Human IgG

+++ (highG1-4)

+++ (moderateG3,4-higG1,2)

Binding properties of protein G

Ligand

Protein G minus albumin binding region

No of IgG binding

2 sites per ligand

Ligand pI

4.1

Binding capacity

>20mg human IgG/ml gel

Matrix

Highly cross-linked agarose , 4%

Ligand density

2mg protein G/1ml gel

Chemical stability

Stable to all commonly used aqueous buffers - 1 M acetic acid, 1% SDS, and 6 M guanidine HCl (tested at 37 C for 7 days), as well as, 0.1 M glycine NaOH pH 11, 1 M HC1, and 8 M urea (stable for at least 2 h at room temperature)

pH stability

3-9 (long term), 2-9 (short term)

Sanitization

Do not autoclave.Wash the column with 70% ethanol

Antimicrobial agent

20% ethanol

Binding capacity (IgG,mg/drained gel, ml)

human
17
rat
7
sheep
18
rabbit
19
goat
19
guinea-pig
17
cow
23
mouse
6


SCHEME
protein G
enlarge  ]
 
REFERENCES
                                                                                                                                      
Nilson, B. H. K. et al (1993) J. Immunol. Meth. 164: 33-40


created by Andrei Musaji          ::          updated : 2004