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Coupl ing Ant ibod y t o CNBr Sepharose

1. Dialyze purified ant ibody against 0 .2 M NaHCO3 , pH 8.9 . Use Spect raPor 2 ( 12 -14 kDal cut of f)

t ubing and tubing clips. The volume of ant ibody solution should decrease during dialysis. Dialyzeovernight at 4 C in at least 10 0 volumes of buff er. Change buff er t hree t imes.

2. Aft er dialysis, determine t he prot ein concent ration (BioRad Dye method). You will need 2-8

mg/ ml final concentrat ion. If needed, concent rate t he ant ibody by Amicon or Centricon filt ration.

Place antibody solution in 15 ml red cap t ube on ice. Save a small amount ( ~1 0 l) of antibody.

3. Wash 10 mls of packed Sepharose 4B with 50 mls ddH2O.

Wash procedure: Use sintered glass funnel and vacuum flask. Resuspend wit hout suct ion. Wait ~1minute. Apply vacuum unt il a dry cake is obt ained. Do not over dry.

4. Wash wit h 50 mls 1M Na2CO3 , pH 11 .

5. Transfer packed Sepharose to a small glass f lask and place on a rot at ing platf orm in a fume hood.

Add 10 mls of 1M Na2

CO3

, pH 11 t o t he flask, washing down walls of t he flask in t he process.

6. Dissolve 1 g of CNBr in 1 ml acetonitrile in 15 ml t ube. Do in fume hood. CNBr is highly t oxic!

7. While swirling the Sepharose suspension, add CNBr solut ion. Swirl f or 10 minut es. Check pH

aft er addit ion of CNBr. Use ColorpHast pH paper (pH 7.5 -14 ) . Add 4 N NaOH t o adjust t he pH t o10 .5-1 1. Add 0.5 ml at a t ime; 2-3 mls may be needed. Check and adjust pH every 2 minutes.

8. Transfer Sepharose to sintered glass funnel in vacuum flask in fume hood. Apply vacuum t o remove

CNBr solut ion. Collect CNBr in 50 mls of 2 % FeSO4 - 7 H2O in vacuum flask.

9 . Washes: 1 X 5 0 mls ddH2O

1 X 10 mls of 9 5% acet one

5 X 50 mls of 0.2 M NaHCO3 , pH 8.9 .

10 . Transfer packed CNBr activat ed Sepharose t o 15 ml tube wit h antibody. Measure amount added

based on increase in volume. Aim for 10 mg ant ibody per ml Sepharose. Minimum is 1 mg/ ml.Mix slowly overnight on rot ator at 4C.

11 . Spin down beads in IEC centrif uge for 5 minutes at 2 00 0 rpm. Save supernat ant on ice.

Det ermine t he eff iciency of coupling by comparing amount of ant ibody in supernatant t o amount insaved aliquot. Ideally, about 7 0-80 % of t he antibody will be bound. Excessive binding (9 0-

10 0%) may reduce ant ibody react ivit y.

12 . Resuspend pellet in ~10 mls of 0 .2 M NaHCO3 , pH 8.9 and t ransfer t o 1 red-cap 50 ml t ube.Rinse all of t he Sepharose into the 50 ml tube. Add 0 .2 M NaHCO3 , pH 8 .9 t o 40 mls. Spin in IEC.

1 3. Washes: 1 X 4 0 mls 0 .2 M NaHCO3 , pH 8.9

1 X 40 mls 0.5 M NaCl, 0.2 M NaHCO3 , pH 8.9 (1 .5 g NaCl/ 50 mls)

1 X 40 mls 0.2 M NaHCO3 , pH 8.9

14 . Quench in 40 mls of 50 mM ethanolamine, pH 7.5. Rot ate at room temperat ure for 4 hours.

15 . Wash beads 3 X PBS. St ore at 4C as 50 % slurry in PBS + 0 .01 % merthiolat e.

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Harlow and Lane Method

Have 20 m g of pure ant ibody in 4-2 0 mls of 50 0 mM NaPi , pH 7.5 in a 50 ml red-cap tube on

ice. The antibody solut ion must be at least 1 mg/ ml. To concentrate ant ibody, use Amicon Filtrationsyst em, or Cent ricon. Dialyze against 5 00 mM NaPi , pH 7.5 . Use Spect raPor 2 (1 2 -14 kDal cut

of f ) t ubing. Use t wice as much dialysis t ubing as needed for t he dialyzed volume. This will allow

enough space for expansion during dialysis. Use tubing clips at t he ends. Dialyze overnight at 4Cin at least 1 00 volumes of buf fer. Change buff er three t imes.

Save a small amount of ant ibody solution to test coupling.

2. Wash 10 mls of packed Sepharose 4B wit h 100 mls ddH2O.

Wash procedure: Use sintered glass funnel and vacuum f lask. Resuspend wit hout suct ion. Wait~1 minut e. Apply vacuum unt il a dry cake is obt ained. Do not over dry.

3. Wash wi th 100 mls 1M Na2CO3 , pH 11 .

4. Resuspend Sepharose in 10 mls of 1M Na2CO3 , pH 11 . Transfer t o a small glass beaker wit h a st ir

bar. Place beaker on stir plat e in a fume hood.

5. Dissolve 1 g of CNBr in 1 ml acet onit rile. Do in fume hood. CNBr is highly t oxic!

6. While t he Sepharose suspension is st irring rapidly, add CNBr solut ion t o t he beads. St ir for 10

minutes. Check pH every 2 minut es (pH paper). If needed, adjust w it h NaOH t o keep pH ~1 1.

7. Transfer Sepharose to sintered glass funnel. Apply vacuum t o remove CNBr solut ion (in FeSO4) .

8 . Wash wit h: 100 mls ddH2O

10 mls of 9 0% acetone

9. Wash wit h 5 X 100 mls of 5 00 mM NaPi , pH 7.5 .

10 . Transfer dry CNBr-Sepharose to 50 ml tube wit h ant ibody. Mix slowly overnight on rot ator at4 C.

11 . Spin down beads in IEC centrif uge for 5 minutes at 2 00 0 rpm. Save supernat ant on ice.

Determine the amount of ant ibody bound by comparing amount in supernatant t o amount in

saved aliquot. Ideally, about 7 0-80 % of t he antibody will be bound. Excessive binding (9 0-10 0%) may reduce ant ibody react ivit y.

12. Resuspend pellet in ~90 mls 50 0 mM NaPi , pH 7.5 and transfer to 2 red-cap 50 ml tubes.

13 . Wash w ith 500 mM NaPi , pH 7 .5. For washes, resuspend pellet s in ~50 mls each. Then, spin

down beads in IEC centrif uge for 5 minutes at 20 00 rpm.

14. Wash wit h 1 M NaCl, 50 mM NaPi , pH 7.5.

15 . Quench by resuspending in 50 m ls of 10 0 mM ethanolamine, pH 7.5 . Rot ate gently in red-cap

tube at room temperature for 4 hours.

16 . Wash beads 3 X PBS. Resuspend beads t o make 50 % slurry using PBS + mert hiolate (1/ 10 0

dilut ion of 1% mert hiolat e).