[ 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
- Slowly (!!!) add the solution of ammonium sulphate
(40-50% final, v/v) to hybridoma cell culture supernatant (60-50%
final, v/v). Store at 4ƒC ,ON (overnight).
- centrifuge the precipitant at 5000-10000 rpm for
10-15 min at RT.
- resuspend the precipitant in PBS (as small volume as
best).
- dialyse against PBS, ON for 48 hours.
- filter on 0.22 µm filter.
- run ELISA to establish the Ig concentration
SOLUTIONS
- ammonium sulphate, saturated, pH 6.8, store at RT
- 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 |
- Use this step
when purifying ascites or serum
- Use SeroClear :
ascitesFluid/serum =1:1,5->vortex
1min,RT->spin 5000rpm,10min->
retain the top layer for subsequent purification
|
- SeroClear- CalBiochem cat. No.437616, not compatible with
polysterene tubes
- 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
- 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 |
- Equilibrate the
column with 5-10 CV(column volums) of 20mM sodium phosphate , pH7
- Dilute ascites or
serum 1:1 with 20mM sodium phosphate , pH7, !FILTER 0,8->0,45-0,22um
and then load onto the column
- Loading rate : 50ml/hr (0,8ml/min)
|
- 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
- alternatively
it`s possible to incubate the gel directly with serum or ascitesFluid
for 30min before applying the gel to
the column
- alternatively
dilute 2-fold for tissue culture SN or 10-fold for ascitesFluid and
bioreactor SN in the loading buffer
|
| Step 2 -
Whashing |
- wash the column
with 20mM sodium phosphate , pH7
- collect the
flow-through
|
- usually
5-10CV are used to wash the column
- check the OD280 of the flow through to
determine when whashing is complete
|
| Step 3 -
Elution |
- elute the bound Abs
with 0.1M glycine-HCl ,
pH 2.7 (2.2)
- collect
fractions and monitor the OD280
- collect fractions
into tubes containing 50-100ul
2M TRIS base , pH8.0per 1ml of fraction , pool fractions
|
- alternatively
use 0,1M acetic acid , pH 2.8 to elute
- Ab
can be stored in 2M TRIS base , pH8.0,or can be exchanged for PBS x 1 or TRIS buffer by dialysis
- Add
0,05% NaN3 if stored at 2-8C and 50% glycerol for -20C
|
| Step 4 -Washing, Cleaning and Gel
storage , Re-equilibration |
- Wash with 0.1M glycine-HCl , pH 2.7
- Clean with C2H5OH 70%-wash , incubate for 12hrs
- Store with 20% C2H5OH, store at 2-8C,
don`t
freeze
- Re-equilibration
with 20mM sodium phosphate , pH7
|
- alternatively
use 1M acetic acid for washing (3-4CV)
- alternatively
use 100mM PBS , pH7.4 with Proclin 0.01% as a preservative for storage
- 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 µm 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 ƒC 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
[ enlarge
]
REFERENCES
Nilson, B.
H. K. et al (1993) J. Immunol. Meth. 164:
33-40
|
|
