Conjugation protocol

Chemicals: Detection antibody

5' aminomodified polynucleotide

TSE pH 8.5 (0.1 M triethanolamine, 0.1 mM

sodium chloride, 1 mM EDTA, pH 8.5)

2-iminothiolane

Glycine pH 7.3

TSE pH 12.9 (0.1 M triethanolamine, 0.1 mM sodium chloride, 1 mM EDTA, pH 12.9) SMCC DMSO

TSE pH 7.3 (0.1 M triethanolamine, 0.1 mM sodium chloride, 1 mM EDTA, pH 7.3)

Figure 12.6

Conjugation of DNA and antibody with SMCC.

Equipment:

Precision pipettes

NAP-5, NAP-10, or PD-10 columns (Amersham

Biosciences)

Centricon YM-30 (Millipore)

Vortex

Centrifuge

Glass vial

1. Change buffer of the detection antibody solution to TSE (pH 8.5) and concentrate to 3-5 mg antibody per ml using, for example, Centricon YM-30 (Millipore)

2. Prepare 50 mM solution of 2-iminothiolane in TSE (pH 12.9) and mix immediately with the antibody solution in proportion 1:33. For example, if the antibody volume is 1 ml, add 30 ^l 2-iminothiolane. Incubate for 30 min at room temperature

3. Quench the reaction with 1 M glycine (pH 7.3) using the same volume as of 2-imino-thiolane

4. Purify the activated antibody with a small gel filtration column such as NAP-5, NAP-10, or PD-10 using TSE (pH 7.3). The activated antibody is not very stable and should be used within 2 h

5. Dilute the polynucleotide to about 70 nmol ml-1 in 0.1 M TS (pH 7.7). The molar amount of the polynucleotide should be approximately two times the molar amount of antibody used

6. Dissolve 3-5 mg of SMCC in DMSO to 20 mM in a small glass vial

7. Add SMCC to the polynucleotide solution to a concentration of 2 mM. Incubate for 20 min at room temperature

8. Quench the reaction by adding 1/50 volume of 1 M glycin (pH 7.3) to the tube

9. Purify the activated polynucleotide with a small gel filtration column, such as NAP-5, NAP-10, or PD-10. Elute with TSE (pH 7.3). The activated polynucleotide should be used within 2 h

10. Determine the concentrations of antibody and polynucleotide spectroscopically

11. Mix the polynucleotide and antibody in a 2:1 molar ratio

12. Incubate 1 h at room temperature, and store the reaction tube in the refrigerator for at least 2 h and at most 72 h before purification

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