Immuno-affinity chromatography
Preparation of antibody-sepharose
Covalently linking an antibody to Sepharose (CL-4B, or CL-2b
for high MW antigen), using CNBr activation method.
1.
dialyze 1-30 m g/ml antibody against 0.1 M NaHCO3 /0.5
M NaCl at 40C with 3 buffer changes over 24 hrs, use
dialysis solutions 500 times of the antibody volumn.
2. Centrifuge
3. Measure A 280 (mg/ml IgG ＝A 280 / 1.44), dilute to 5
mg/ml with 0.1 M NaHCO3 /0.5 M NaCl
4. Wash the sepharose with 10 vol water, use Waterman no. 1
filter and Buchner funnel.
5. Add equal volumn of 0.2 M Na2CO3 to Sepharose.
6. Add CNBr/ acetonitrile dropwise (in hood).
7. Filter, dry, and add 0.1 M HCl, and add antibody solution, for
2 hr at rt.
8. Add glycine to saturate the active group on Sepharose, and
measure A 280 to determine percentage coupling
Immuno-affinity chromatography
1. Pack Ab-sepharose in one column, as affinity column
2. Pack activated, quenched sepharose (prepared as for Absepharose but eliminating Ab or substituting irrelevant Ab
during coupling) in the other column, as pre-column.
3. The two columns are connected in parellel.
4. Wash columnes, with 10 volume of wash buffer
(containing Tris, Triton-X-100,
0.14 M NaCl,
0.5% Na deoxycholate, pH 8).
5 vol. of Tri/Triton/NaCl buffer, pH 8
5 vol. of Tri/Triton/NaCl buffer, pH 9
5 vol. of Triethanolamine solution
5 vol. of wash buffer
5. Apply protein sample, flow rate: 5 vol/hr.
6. Collect flow through fraction, each 1/10 to 1/100 vol. of
applied sample.
7. Wash with 5 vol. of wash buffer
8. Wash the affinity column only with 5 vol. of wash buffer
5 vol. of Tri/Triton/NaCl buffer, pH 8
5 vol. of Tri/Triton/NaCl buffer, pH 9
9. Elute antigen with 5 vol. Triethanolamine solution ( pH may
varied with different antigen)
10. Collect 1 vol to tube containg 0.2 vol of 1M Tris (pH 6.7)
for neutralization/
11. Wash columes with 5 vol. of Tri/saline/azide solution and
storte colume at 40C.
12. Analyze antigen infraction by immunoprecipitation with Absepharose.
Low pH elution of antigen protocol, (in cases antigen is not
stable in alkaline condition),
protocol for batch adsorption and elution in column, and
protocol for obtaining postnuclear supernatant, are provided.
Metal-Chelate Affinity Chromatography
(MCAC)
For soluble His-tag proteins
1. Grow BL21(DE3) pLys / pET vector that express His-tag
protein in M9ZB/Ap/Cm broth, overnight.
2. Inoculate 1 ml to 100 ml M9ZB/Ap/Cm broth, grow to OD600
= 0.7 to 1.0.
3. Add IPTG to 1mM final, and incubate for 1-3 hr (less
induction time means less recombinant protein, but likely
more soluble).
4. Centrifuge, and freeze pellet and thaw at 40C, or go to 6.
immediately.
5. (While centrifugation, add 0.2 ml NTA resin to an empty
column, and wash with 1 ml dH2O
1 ml of NiSO4.6H2O
2 ml MCAC-0 buffer
Charged resins are light-blue uncharged are while)
(For regeneration NTA resin, Wash resin with acetic acid/
Guanidine, water, 25%, 50%, 75%, 100%, 75%, 50%, 25%
EtOH, water, EDTA, water, stepwise, then recharge as
above )
6. Add 5 ml MCAC-0 buffer and protease inhibitor cocktail
(sigma), and pipetting, sonication, or homogenization.
7. Add Triton X-100 (0.1% final), and 3 cycles of freeze -thaw.
8. Add MgCl2 and DNaseI, incubate at rt for 10 min.
9. Centrifuge, and keep supernatant, and store indefinitely at 700C (thaw on ice before use). Save 10 μl for SDS-PAGE.
10. Load extract on column (5-10 mg his-protein / ml packed
resin), flow rate 10-15 ml/hr. collect flowthrough for SDSPAGE.
11. Wash with MCAC-0 at 20-30 ml/hr.
12. Wash with 5 ml each of MCAC-20, MCAC-40, MCAC-60,
MCAC-80, MCAC-100, MCAC-200, MCAC-1000 at 10-15
ml/hr. Collect 0.5 ml fractions for SDS-PAGE.
(The 2nd and 3rd fractions of each wash contains most
proteins.
Most His-proteins elute at MCAC-100 and MCAC-200.)
13. Elute colume with 1ml MCAC-EDTA buffer.
14. Analyze presence of proteins by SDFS-PAGE.
15. Pool the fractions with the recombinant proteins. . If
desired, dialyze against buffer. .
16. Aliquot, and store at -70C or liquid N2.
MCAC-0: 20 mM Tris, pH 7.9
0.5M NaCl
10%glycerol
1 mM PMSF
MCAC-20: MCAC-0 with 20mM imidazole.
For Insoluble His-tag proteins
5. wash charged column with 2 ml GuMCAC-0 buffer.
6. Thaw cell pellet on ice and resuspend in 5 ml
GuMCAC-0 buffer. and pipetting, sonication, or
homogenization.
7. Freeze at -700C and thaw at rt.
8. Load on column.
9. Wash column with GuMCAC-20, -40, -60, -80, -100, 500 buffer at 10-15 ml/hr. Collect 0.5 ml fractions for
SDS-PAGE.
10. Analyze and pool the desired fraction.
11. Bring to final 4M guanidine. Dialyze first against buffer/
4M guanidine, then buffer/ 2M guanidine, then buffer
only.
12. Aliquot, and store at -700C or liquid N2.
For solid-phase renaturation of MCAC-purified proteins
After step 8,
1. Wash column with 5 ml 1:1 (V/V) MCAC-20/ GuMCAC-20
3:1
7:1
(or linear elution from 100% GuMCAC-20 to MCAC-20 in 1-2
hrs.)
2. Wash with MAC-0 buffer, elute proteins.
HPLC of peptides and proteins
- Preparation of samples:
Dissolve sample with half the target volume of eluent
A (weak mobile phase).
If not soluble, add <25% of total volume of eluent B
(strong mobile phase).
Pass through 0.2 μM PTFE filter, and store at -200C.
- Preparation of mobile phase: Mix well, and pass through
0.2 μM PTFE filter.
-Detection:
Peptide bonds absorb at far UV region (205 -215 nm),
Usually used 215 nm for compromise with buffer adsorption.
Eight modes for proteins and peptides
1. Size-exclusion chromatography (HP-SEC):
proteins activity retained.
2. Normal-phase chromatography
3. Hydrophobic interaction chromatography
4. Ion-exchange chromatography
5. Reversed phase chromatography (RP-HPLC):
used mostly often.
6. Hydrophilic interaction chromatography
7. immobilized metal ion affinity chromatography
8. biospecific/ biomimetic affinity chromatography
Size-exclusion chromatography (HP-SEC)
eg.
Column: T-250 (10 mm, 300 Å, 300-mm length x
7.5-mm i.d.)
Sample size: <2 mg peptide/protein
Sample loop size: 20 to 200ml
Isocratic elution
Eluent A: 50 mM KH2PO4, pH 6.5, 0.1 M KCl
Flow rate: 0.5 ml/min
Detection: 214 nm
Temperature: room temperature
Reversed phase chromatography (RP-HPLC)
I. For peptide analysis
eg.
column: e.g., C4, C8, C18 (5 mm, 300 Å, 150 -mm length x
4.6-mm i.d.)
Sample size: <2 mg peptide/protein
Sample loop size: 20 to 200ml
Liner A→B gradient
Eluent A: 0.9% aquous TFA (trifluoroacetic acid)
Eluent B: 0.1% TFA in acetonitrile/water
Gradient rate :1% B/min (eg. 60% acetonitrile/water in
60min)
Flow rate: 1.0 ml/min
Detection: 214 nm
Temperature: room temperature
II. For peptide purification
eg.
column: C4, C8, C18 (10 mm, 300 Å, 300-mm length x
21.5-mm i.d.)
Sample size: <150 mg peptide/protein
Sample loop size: 1 ml, multiple injection
Liner A→B gradient
Eluent A: 0.9% aquous TFA
Eluent B: 0.1% TFA in acetonitrile/water
Gradient rate: 0.66% B/min (eg. 60% acetonitrile/water
in 90min)
Flow rate: 7.5 ml/min
Detection: 254 nm (sacrifice 1mg protein to scan for best
wavelength)
Temperature: room temperature
III. For desalting of protein and peptide solutions
(Fig. 10.12.9)
eg.
Column: e.g., C4, C8, C18 (10 mm, 300 Å, 300-mm length x
4.6-mm i.d.)
Sample size: 8mg peptide/protein
Sample loop size:1ml
Step elution
Eluent A: 0.1% aquous TFA
Eluent B: 0.1% TFA in acetonitrile or 2-propanol
Elution condition: 100% eluent A for 3 min, then 100%
eluent B for 3 min.
Flow rate: 2.5 ml/min
Detection: 230 nm
Temperature: room temperature
Immunoprecipitation
Immunoprecipitation with recombinant proteins
1. Sample, Protein A or protein G-agarose beads, and normal
mouse IgG, incubate
2. centrifuge
3. Collect supernatant, add epitope-specific Ab, 40C for 3-4 hr.
4. centrifuge
5. wash pellet and add SDS-sample buffer.
6. Boil, centrifuge, and analyze supernatant on
SDS-PAGE.