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, fo

r 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 column2. Pack activated, quenched sepharose (prepared as for Ab-se

pharose 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 buffer5. Apply protein sample, flow rate: 5 vol/hr. 6. Collect flow through fraction, each 1/10 to 1/100 vol. of appli

ed sample.

7. Wash with 5 vol. of wash buffer8. 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 99. 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) fo

r neutralization/ 11. Wash columes with 5 vol. of Tri/saline/azide solution and s

torte colume at 40C. 12. Analyze antigen infraction by immunoprecipitation with Ab-

sepharose.

Low pH elution of antigen protocol, (in cases antigen is not stable in alkaline condition),

protocol for batch adsorption and elution in column, andprotocol for obtaining postnuclear supernatant, are provided.

Metal-Chelate Affinity Chromatography (MCAC)

For soluble His-tag proteins1. Grow BL21(DE3) pLys / pET vector that express His-tag prot

ein 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 inductio

n 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/ G

uanidine, 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 SDS-PAGE.

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 homog

enization. 7. Freeze at -700C and thaw at rt. 8. Load on column.9. Wash column with GuMCAC-20, -40, -60, -80, -100, -50

0 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 chromatography3. Hydrophobic interaction chromatography4. Ion-exchange chromatography5. Reversed phase chromatography (RP-HPLC): used mostly often.6. Hydrophilic interaction chromatography7. immobilized metal ion affinity chromatography8. 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. centrifuge3. Collect supernatant, add epitope-specific Ab, 40C for 3-4 hr.4. centrifuge5. wash pellet and add SDS-sample buffer.6. Boil, centrifuge, and analyze supernatant on SDS-PAGE.