Materials

1. Poly- or monoclonal antibody or antibody fragments (see Note 1).

2. Reactive biotin derivative (see Note 2 and Tables 1-3). These reagents are available from several companies, including Molecular Probes, Inc., Eugene, OR; Pierce Chemical Co., Rockford, IL; and Sigma/Aldrich, St. Louis, MO. Three kits containing all of the components necessary (except buffers) to biotinylate or desthiobio-tinylate antibodies (and other proteins) are available from Molecular Probes, Inc. The FluoReporter® Mini-Biotin-XX Protein Labeling Kit (F-6347) contains sufficient reagents for five biotinylations of 0.1-3 mg each of antibody. The FluoReporter® Biotin-XX Labeling Kit (F-2610) is formulated for researchers who want to biotinylate five or more 5-20 mg amounts of protein and determine their degrees of labeling. The DSB-X™ Biotin Protein Labeling Kit (D-20655) is designed for five desthiobiotinylations with 0.5-3 mg each of an antibody or another protein.

3. Reaction buffer: 100 ml bicarbonate buffer (pH 8.0-8.3) is preferred for labeling antibody amines (see Note 3). The optimum reaction pH values for labeling antibodies with other reactive biotin derivatives are summarized in Table 3.

4. Other necessary materials:

a. High-quality, heavy metal-free, well-washed dialysis tubing (e.g., Spectrum Laboratories Inc., Compton, CA or b. Gel filtration (size-exclusion) chromatography matrix and column (see Note 4).

Table 1

Structures of Reactive Biotin Derivatives

i ch3

hKr' a 2-Iminobiotin i-NH Desthiobiotin

R

Name Reactivity

Available attached to

-O—N

SE Amines

- NH-(CH2)5—C—0— N 0

X-SE Amines

— NH-(CH,)5— C — NH(CH2)^C-

-0-N

XX-SE Amines

B

— NH-(CH2)5—C— 0— N

^SOi

X-SSE Amines

- NH-(CH2)5— C — NH(CH2)5— C —

K^SOi

D— N

XX-SSE Amines

- NH-(CH2)5— C—NHNH2

X-hydrazide Aldehydes

B,D

— NH-(CH2)5—C— NH-(CH2)5— C — NHNH2

XX-hydrazide Aldehydes

- NH-(CH2)6-NH—C-CH2-

-

Iodoacetyl Thiols

B

- NH-(CH2)6-NH—c- (CH2)2—s-s~C°)

HPDP Thiols

B

- NH—(CH2)4—NH — C—^

)-CH2— N^J) o

Maleimide Thiols

B

— NH—(CH2)3—N (CH2)3—

no2

Photoactivatable Nonspecific

Structures of Reactive Biocytin Derivatives

Table 2

Structures of Reactive Biocytin Derivatives

<ch2)4 Biocytin

<ch2)4 Biocytin

R

Name

-CH(NH2)-C-OH

Biocytin

— CH(NH,)—C — NHNH2

Hydrazide

Aldehydes

0 ° —CH(COOH)—NH—C—(CH2)2—N j) 0

Maleimide

Summary of Recommended Conditions for Reactive Biotin Derivatives

Table 3

Summary of Recommended Conditions for Reactive Biotin Derivatives

Reactive group

MW"

Reaction pH

MR for IgG (at 10 mg/mL)

SE

341.38

8.0-8.3

10-20fe

X-SE

454.54

8.0-8.3

10-20fe

XX-SE

567.70

8.0-8.3

10-20fe

X-SSE

556.58

8.0-8.3

10-20fe

XX-SSE

669.74

8.0-8.3

10-20fe

X-hydrazide

371.50

6.0-7.2

20-25

XX-hydrazide

484.66

6.0-7.2

20-25

Iodoacetamide

510.42

7.5-8.0

16-20

HPDP

539.77

6.0-9.0

16-20

Maleimide

533.69

6.5-7.2

16-20

Photoactivatable

533.65c

7.0-7.5

5

"When the reactive group is attached to biotin.

6For IgG at <1 mg/mL (30-40), 1-3 mg/mL (20-25), 4-10 mg/mL (10-20). cAs sold by Pierce Chemical Company (Rockford, IL).

"When the reactive group is attached to biotin.

6For IgG at <1 mg/mL (30-40), 1-3 mg/mL (20-25), 4-10 mg/mL (10-20). cAs sold by Pierce Chemical Company (Rockford, IL).

c. Molecular weight-selective, centrifugable concentration devices (see Note 5).

d. Anhydrous DMSO or DMF (see Note 6). 5. Optional materials:

a. Sodium cyanoborohydride, available from Sigma/Aldrich and other vendors.

b. HABA (4'-hydroxyazobenzene-2-carboxylic acid), available from Pierce Chemical Co., Sigma/Aldrich, and other vendors.

c. Avidin or streptavidin, available from Molecular Probes, Inc. (A-887, S-888), Pierce Chemical Co., Sigma/Aldrich, and other vendors.

d. D-biotin, available from Molecular Probes, Inc. (cat. nos. B-1595, B-20656), Pierce Chemical Co., Sigma/Aldrich, and other vendors.

0 0

Post a comment