AFFINITY PURIFICATION OF ANTIBODIES
作者：佚名
实验频道来源：本站原创
点击数： 85
更新时间：2004-8-3
AFFINITY PURIFICATION OF ANTIBODIES
(Fisher et al.,1988, Cell 54: 813-822)
Conjugation of carrier (BSA) to peptide
1. Dissolve pure BSA @ 20mg/ml in 10mM phosphate, pH 7.0.
2. Dissolve MBS-SMCC [m-maleimidobenzoyl N-hydroxysuccinimide ester succinimidyl 4- (N-maleimidomethyl)cyclohexane
-1-carboxylate] @ 25mg/ml in NNdimethylformamide.
3. Mix 20ml SMCC and 200ml BSA (500mg:4000mg or 1:8); incubate ON @ RT.
4. Separate conjugate from unreacted crosslinker on a G25 column in 100mM phospate, pH 6.2. Identify the peak by A
280 and pool peak (1 ml) fractions.
5. Dissolve peptide (1-5 mg)in 1ml 0.1M Borate, pH 9.1.
Reduce by adding 100ml 0.1M NaBorohydride (NaBH4), mix.
Let stand 10'.
Lower pH with 70ml 1M HCl to inactivate NaBH4.
Neutralize with 40ml 1N NaOH.
6. Combine carrier and peptide and mix gently ON.
7. Fractionate over G25 ON in 0.1X PBS, A280, pool peak and lyophilize.
8. Check peptide coupling (1mg samples) on a Laemmli gel.
Affinity Column Prep
1. Affigel 102 (BIORAD) washed in PBS on a scintered glass funnel (3ml/column).
2. Transfer to 15ml screw cap tube in minimal volume.
3. Add 7mg SMCC (in DMF) to 2.5ml beads and mix for 2h @ RT.
4. Wash beads again in PBS.
5. Resuspend in minimal volume 100mM phosphate, pH 6.2.
6. Add BSA-peptide and react ON with mixing.
7. Wash beads extensively.
8. Pack into a 3ml syringe as column. Add azide!
Affinity Purification
1. Equilibrate column with PBS, pH 7.4.
2. Load on sample diluted (1:10 ?)to run slowly ON (e.g., 160ml @ 8ml/h = 20h).
3. Rinse column: 50ml 2M NaCl, pH 7.5
50ml 0.1M Borate, pH 9.1
50ml 0.1M PBS, pH 4.5
4. Elute antibody with 20mM glycine-HCl/200mM NaCl. Collect 0.8ml fractions into tubes containing 0.4ml 0.1M Tris-HCl,
pH 8.5.
5. Assay fractions by A280 and pool peak. Store some @ 4oC (+0.1% NaN3) and freeze remainder @ -70oC.
acc 12/90
Antibody Purification
This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinit
y chromatography.
Solutions
Affigel Blue Prewash
0.1 M acetic acid 5.7 ml glacial acetic acid
1.4 M NaCl 81 g NaCl
40% isopropanol 400 ml isopropanol
up to 1 liter with Q
check to make sure pH is 3.0
Affigel Blue Running Buffer
10 mM K2HPO4 2.28 g K2HPO4
0.15 M NaCl 8.2 g NaCl
0.02% azide 0.2 g Na azide
up to 1 liter with Q
1.4 M NaCl
8.1 g NaCl
up to 100 ml with Q
Saturated NH4SO4
767 g NH4SO4
add 1 liter Q
10X PBS
80 g NaCl
2 g KCl
14.4 g Na2HPO4
2.4 g KH2PO4
up to 1 liter with Q
Affigel Blue Regeneration Buffer
2 M guanidine HCl or
1.5 M Na thiocyanate
HiTrap Storage Buffer
10 mM Tris 7.5 1 ml 1M Tris 7.5
0.1 mM EDTA 20 ul 500 mM EDTA
50 mM NaCl 1 ml 5M NaCl
0.1% azide 0.1 g Na azide
up to 100 ml with Q
1X Coupling Buffer
0.2 M NaHCO3 1.68 g NaHCO3
0.5 M NaCl 2.92 g NaCl
pH to 8.0 and bring up to 100 ml
1X Buffer A
0.5 M NaCl 2.92 g NaCl
0.01 M Tris 0.121 g Tris base
pH to 8.3 and bring up to 100 ml
Add 3.0 ml ethanolamine before use
1X Buffer A
0.1 M NaOAc 0.82 g NaOAc
0.5 M NaCl 2.92 g NaCl
pH to 4.0 and bring up to 100 ml
Procedure
• Pour a 5 ml affigel blue column (biorad) and wash with 50 ml Prewash to prep the c
olumn (when first used or if last used in more than a week).
• Wash with 50 ml Q, followed by 50 ml Running Buffer.
• Wash with 50 ml 1.4 M NaCl, if eluate is colored, then re-equilibrate.
• Wash with 50 ml Running Buffer.
• Load 1 ml serum, save flow-through, elute with 2 bed volumes running buffer (the se
rum albumin should stick to the column and the Ig should flow through).
• On ice slowly add saturated ammonium sulfate to 45% (550 ml sample + 450 ml sat
urated ammonium sulfate). Tilt overnight at 4°C.
• Pellet by spinning at 3,000 rpm for 10 minutes at 4°C.
• Resuspend the pellet in 1 ml 1X PBS on ice (don’t vortex or agitate) and dialyze ag
ainst PBS.
• The pharmacia HiTrap 1ml column can bind 10 micromoles of peptide or protein per
ml of bed volume. Prep the HiTrap column by washing with 10 ml 50% Isopropanol, 2
5% Isopropanol, 10% Isopropanol, and 10 ml 1 mM ice cold HCl.
• Resuspend the peptide or protein in 1 ml 1X Coupling Buffer and load the column. H
old at room temperature for 1 hour.
• Wash with 5 ml 1X Coupling Buffer and save the flowthrough.
• Wash with 6 ml Buffer A, 6 ml Buffer B, and 6 ml Buffer A. Hold at room temperatur
e for 30 minutes.
• Wash with 6 ml Buffer B, 6 ml Buffer A, and 6 ml Buffer B. Wash with Storage Buff
er and hold at 4°C.
• To purify the antibody, load the affigel concentrate onto the column and hold at room
temperature for 10 minutes.
• Wash with 50 ml 10 mM Tris 7.5 and then wash with 50 ml 10 mM Tris 7.5/500 mM
NaCl. Elute with 5 ml 100 mM Glycine pH 2.5 into 1 ml 1M Tris 8.0.
• Dialyze and concentrate with a centricon 30
Purification of mAb (IgG)
作者：佚名
实验频道来源：本站原创
点击数： 299
更新时间：2004-8-3
Purification of mAb (IgG)
by Chang-Duk Jun, 03/14/2000
Purpose
Materials
o
Antibody 7E3, 2L sup grown in flasks, frozen and thawed overnight.
o
BioRad Affi-Gel Protein A MAPS II Buffers cat. #1530-6160 ($161.00)
o
50 mM Tris pH7.8
o
40 g ammonium sulfate for every 100 ml Sup.
o
4L 100 mM Tris/pH7.8 or Binding Buffer
o
BD tubing 14-170-12F (Fisher)
o
Polyethylene tubing Clay Adanes.
o
18 Gauge needles.
o
Binding Buffer (BioRad): make 1L. 314g/L ddH2O and filter through 0.22 um, filter. pH=9.0
o
Elution buffer (BioRad): make 500 ml. 11g/500 ml ddH2O and filter as before. pH=3.0
o
Regeneration buffer (BioRad): BioRad Affi-gel regeneration buffer.
Procedure
For Ammonium sulfate cut:
1.
Filter Sup. in 1L Costar Filter using pre-filter.
2.
Add 50 mM Tris pH7.8 (50 ml of 1 M stock/Liter). 100ml
3.
Add 40 g ammonium sulfate for every 100 ml Sup. (slowly). 800g
4.
Stir O/N in cold room.
5.
Pour into plastic bottles. Spin at 4 oC, 7,500 rpm for 20 min in JA-10 rotor.
6.
Prepare 4L 100 mM Tris/pH7.8 (400 ml of 1 M stock/4 L) or Binding buffer.
7.
Discard Sup. Resuspend pellets in 10 ml of 100 mM Tris buffer or Binding buffer. And pool into 50 ml Falcon t
ubes (try not to make bubbles).
8.
9.
Use 3.2 ml/cm 12-14,000 MW dialysis tubing.
o
Heat tubing in 500 ml H2O in microwave. Not boiling.
o
Rinse tubing in fresh H2O several times.
o
Test each tube w/H2O and discard.
Add protein mixture to dialysis tube. Stir slowly in 100 mM Tris buffer or Binding buffer until pink color is out.
10. Transfer protein mixture (Ab) to 50 ml Falcon tubes to determine volume.
11. Dilute the mixture 1:1 with Binding buffer.
12. Filter through 0.45 um filters (use prefilters).
For Purification of Abs by Protein A column:
1.
Prepare Protein A column.
2.
Run binding buffer (pH9.0) ~ 200 ml.
3.
Filter protein mixture
4.
Add protein mixture or culture supernatant containing Ab (adjust pH to 7.8 with Binding buffer; red color) to the
Protein A column.
Mouse antibodies of the IgG1 subclass do not have a high affinity for protein A. Purification on protein A beads
using standard conditions will yield approximately 1/10 the amount of antibody compared with other subclasses.
In case of IgG1 subclass, add 3.3 M NaCl (192.85 g/L) to crude antibody preparation (serum, tissue culture su
pernatant, or ascites).
5.
Apply Binding buffer again ~ 200 ml.
6.
Apply Elution buffer ~ 100 ml.
7.
Collect 3 ml fractions in 5 ml tubes with 700 ul 1 M Tris pH 9.0 already in bottom of tube to neutralize (collect
at ~5 min/fraction).
8.
25 tubes are sufficient for collection. In general, you can see high Ab concentrations in 7-8 tubes.
9.
Test 1 ul on pH paper.
10. Read OD to know Ab concentrations.
OD (Absorbance at 280 nm)/1.35 = X mg/ml. Purification of Antiserum or Ascites by Protein A/G Chromatograph
y
Required Materials and Equipment

Protein A or G agarose gel column (10 ml or 5 ml of packed beads; see guidelines below for choice of protein
A or protein G)

Ice-cold Tris-buffered saline (TBS). See recipe below.

5% sodium azide solution

Neutralization Buffer (NB). See recipe below.

Elution Buffers (pH 2.7 and pH 1.9). See recipes below.

Centrifuge tubes and microcentrifuge tubes

Preparative centrifuge and microcentrifuge

pH strips

Microtiter plate reader with 600 nm filter

Glass Columns
Guidelines for Choosing Protein A Agarose or Protein G Agarose
Antibodies bind with different affinities to protein A and protein G conjugated agaroses. Use the chart below to choose
the best affinity agarose for your antibody
Species and Type of Antibody
Agarose
Rabbit IgG
Protein A or Protein G
Mouse IgG1
Protein G
Mouse IgG2
Protein A or Protein G
Mouse IgG3
Protein G
Sheep IgG
Protein G but binds weakly
Rat IgG
Protein G but binds weakly
Guinea Pig IgG
Protein A
Dog IgG
Protein A
Goat IgG
Protein G
Pig IgG
Protein A
Buffer Preparation
To prepare 1 liter of TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% sodium azide) add the following to 800ml
of distilled water:

6.06 g of Tris base

8.77 g of NaCl

10ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or
function.)
Mix well and adjust the pH to 7.4 with 5 N HCl. Bring the volume up to 1 L with distilled water and recheck the pH a
fter chilling on ice.
To prepare 100 ml of NB (1 M Tris-HCl, pH 8.0; 1.5 M NaCl; 1mM EDTA; 0.5% sodium azide), add the following to
80 ml of distilled water:

12.1 g of Tris base

8.7 g of NaCl

200 microliters of 0.5M EDTA

10 ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response o
r function.)
Mix thoroughly and adjust the pH to 8.0 with 5 N HCl. Add distilled water to obtain a final volume of 100 ml. The Na
Cl and EDTA are added to prevent aggregation of the antibodies and loss of biological activity.
To prepare 100 ml of Elution Buffer pH 2.7 (50 mM Glycine-HCl, pH 2.7), add the following to 80 ml of distilled wat
er:

0.38 g of Glycine
Mix thoroughly and adjust the pH to 2.7 with 5N HCl. Add distilled water to a final volume of 100ml.
To prepare 100 ml of Elution Buffer pH 1.9 (50 mM Glycine-HCl, pH 1.9), add the following to 80 ml of distilled wat
er:

0.38 g of Glycine
Mix thoroughly and adjust the pH to 1.9 with 5 N HCl. Add distilled water to a final volume of 100 ml.
Procedure
Section I: Preparation of a Protein A Agarose or Protein G Agarose Affinity Column
Typically,columns containing 5 ml or 10 ml of packed protein A/G Agarose are prepared. The size of the column is det
ermined by the binding capacity of protein A/G and the amount of antiserum that must be processed. Protein A and pr
otein G bind about 20 mg of IgG per ml of conjugated agarose. Therefore, the binding capacity of a 10 ml (5 ml) colu
mn is 200 mg (100 mg) of IgG. A high-titer rabbit antiserum has roughly 5 mg/ml of IgG, mouse ascites has roughly 1
0 mg/ml of IgG, and goat or sheep antiserum has roughly 20 mg/ml of IgG. Use the guidelines below to choose a col
umn size to avoid exceeding the capacity of the column. Do not exceed 90% of the binding capacity of the colum
n.
10 ml Bead Volume Protein A/G Affinity Column
Source of Antibody
Concentration Volume for Capacity Volume for 90% Capacity
Rabbit Antiserum
5 mg/ml
40 ml
36 ml
Mouse Ascites
10 mg/ml
20 ml
18 ml
Goat/Sheep Antiserum 20 mg/ml
10 ml
9 ml
5 ml Bead Volume Protein A/G Affinity Column
Volume
Source of Antibody
Concentration
for
90%
Volume for Capacity
Capacity
Rabbit Antiserum
5 mg/ml
20 ml
18 ml
Mouse Ascites
10 mg/ml
10 ml
9 ml
5 ml
4.5 ml
Goat/Sheep Antiserum 20 mg/ml
Pouring the Protein A/G Affinity Column
1. Use a pipet to transfer the desired volume of a 50% protein A/G agarose slurry to a vacuum flask. Remember to tr
ansfer enough slurry to prepare a column with the desired bed volume and capacity.
2. Place a stopper in the vacuum flask and apply vacuum for at least 15 minutes at room temperature. This step remo
ves gas from the agarose and is necessary to prevent bubble formation in the column that would reduce the column's
capacity and resolution.
3. While degassing the agarose, chill the TBS on ice. Prepare TBS without azide for antibodies that will be used in viv
o or in cellular assays. TBS is used to equilibrate and wash the protein A/G column.
4. Slowly add the degassed protein A/G agarose to a glass column, e.g., a Biorad Econo Column, using a wide bore
pipet to prevent rupturing the beads.
5. Pack the column at a flow rate of about 1-2 ml per min. Do not let the column run dry! Flow rate may be control
led by using a Mariotte flask or pump.
6. Wash the column with 10 bed volumes of ice-cold TBS. This step chills the column, which reduces nonspecific bindi
ng of proteins and slows the metabolism of any remaining viable microbes.
Section II. Preparation of Antiserum or Ascites for Affinity Chromatography
Throughout this section, "antiserum" refers to antiserum or ascites.
1. Thaw the antiserum in ice water or the refrigerator overnight to prevent aggregation of proteins. Any protein aggregat
e that forms during thawing may be dissolved by briefly warming the thawed antiserum to 37
.
2. Add sodium azide to a final concentration of 0.05%, which is a 1:100 dilution of a 5% stock solution.
CAUTION: Since sodium azide is toxic, wear gloves and handle the stock solution with care.
3. Clarify the antiserum by centrifugation at 15,000 x g for 5 minutes at 4
. This step is performed to sediment aggre
gates of denatured protein and lipid; it is an important step because it removes material that can foul and block the col
umn.
4. Remove the clarified antiserum that lies between the floating lipid and the pellet. Additional filtration may be required
to remove residual lipid.
Section III. Affinity Chromatography Using Protein A/G Agarose
1. Save a 500 microliter sample of the clarified antiserum for testing along side the purified IgG because the a
ntibody may be acid labile.
2. Add the clarified antiserum to the column at a flow rate of 2 ml per minute. (Calibrate the flow rate using a 15 ml c
onical centrifuge tube as the receiving vessel. Adjust the stopcock on the column to give a flow of 2 ml per minute. D
o not exceed a flow rate of 2 ml per minute.) Pass the antiserum through the column twice and save the flow thro
ugh in case the antibody did not bind to the column.
3. Wash the column with a volume of TBS equal to 10 fold the volume of antiserum loaded on the column. For examp
le, if 30 ml of antiserum were loaded on the column, wash with 300 ml of PBS. Save the wash in case the antibody
was eluted.
4. After washing the column, collect a 20 microliter fraction and test for protein by adding it to 180 microliters of Coom
assie Blue reagent to test for protein. If the sample turns blue, wash the column with an additional 50 ml of TBS and
repeat the analysis for protein.
5. Prepare 1.5 ml microcentrifuge tubes for the collection of eluted antibody. Obtain one tube for each ml of antiserum
plus five extra 1.5 ml tubes. Add 100 microliters of Neutralization Buffer (NB) to each tube. Note that IgG may be elut
ed from protein A/G agarose by a change in temperature, ionic strength, and/or pH. All of these conditions affect the bi
nding between the charged amino acids of protein A/G and the Fc region of the IgG heavy chain. When eluting with a
cidic buffer, the acid must be neutralized as quickly as possible to prevent denaturation of the IgG.
6. Once the column has been washed, drain the TBS from the column bed to avoid unnecessary dilution of the Elution
Buffer. Do not let the column stand dry.
7. Gently add Elution Buffer pH 2.7 at room temperature to the column. Be careful not to disrupt the column bed. Use
15 ml of Elution Buffer pH 2.7 if less than 20 ml of antiserum was loaded. Use 20 ml of Elution Buffer pH 2.7 if 2030 ml of antiserum was loaded. Use 25 ml of Elution Buffer pH 2.7 if 30-36 ml of antiserum was loaded. Note that the
se volumes are guidelines and Elution Buffer muse be added until all protein has been eluted from the column.
8. Collect roughly 1 ml fractions in the tubes prepared in step 5 above. Mix each fraction immediately and place on
ice before collecting the next fraction. Neutralizing the acid pH prevents denaturation of the antibodies and loss of
biological activity.
9. Add 10 ml of Elution Buffer pH 1.9 at room temperature to the column; be careful not to disrupt the column bed. C
ollect, mix, and save fractions as described in step 8 above.
10. Remove a 20 microliter sample of each fraction and add to 180 microliters of Coomassie Blue reagent in a microtit
er plate to monitor protein elution. Continue to collect fractions until the color drops to or just below background.
11. Read the microtiter plate at 600 nm and combine all fractions with an absorbance greater than or equal to 0.2 abo
ve background at 600 nm. For example, if the background is 0.18, combine all fractions with an absorbance greater th
an 0.38.
12. Once the absorbance has dropped below 0.2, wash the column with 200 ml of ice-cold TBS with 0.05% sodium az
ide.
13. Store the column at 2-8
.
14. Use pH paper to check the pH of the combined fractions. If the pH is less than 7.0, use NB to adjust the pH to a
pproximately 7.4.
15. Use the Bradford Assay with IgG as a standard, to determine the concentration of the antibody in the combined fra
ctions. Add glycerol to 10% of the total volume if the antibody concentration of the combined fractions is less than 0.5
mg/ml.
16. Store the purified antibody at 2-8
11.
12.
13. Store Abs at -20oC or further concentrate by using Centriprep. And store at -20oC.
14. Regenerate Ab column with Regenerate buffer (~200 ml).
Protein G Purification of Antibodies
Source: Contributed by Nanci Donacki
Reagent and Materials
1. Hi-Trap Protein G Column (Pharmacia Biotech #17-0404-01)
2. 20 mM Sodium Phosphate Buffer, pH 7.0
1.084 g
NaH2PO4, anhydrous
3.273 g
Na2HPO4.7H2O
q.s. to 1 liter with di-H2O
3. 0.1M Glycine, pH 2.8
3.75 g Glycine
1.4 ml HCl, concentrated
q.s. to 1 liter with di-H2O
4. 1M Tris
141.1 g
Tris base
q.s. to 1 liter with di-H2O.
5. 20% Ethanol
Procedure
1. Prepare the collection tubes by adding 0.1 ml of 1M Tris per ml of each fraction to b
2. Centrifuge or filter the sample to remove any particulates.
Adjust to pH 7.0.
3. Wash the column with 5 bed volumes of 20 mM Sodium Phosphate Buffer, pH 7.0.
4. Apply the sample onto the column.
5. Was with 5 bed volumes of 20 mM Phosphate Buffer, pH 7.0
6. Elute the antibody with 1-3 bed volumes of 0.1M Glycine, collecting fractions into tube
1M Tris.
7. Regenerate column with 2-% Ethanol and store at 2-8oC.
8. Pool fractions containing antibody and dialyze against 3 changes of PBS, at least 10
sample volume.
9. Determine the protein concentration of the antibody.
at -20oC.
Concentrate if necessary. St