Supplementary 1: Click chemistry antibody-DNA conjugation
general information and procedure for materials process.
1. Pre-conjugation antibody considerations:
A. Antibody buffer exchange: Commercially supply liquid form of
antibody most commonly contains different types of additives like;
glycerol, NaCl, KCl, potassium phosphate, chelating agents (EDTA,
EGTA) and sugars has no effect on conjugation. But sodium azide has
direct effect on conjugation performances and for standard conjugation
reaction best to avoid any kinds of additives in buffer for the conjugation
reaction. Antibody for 1xPBS buffer exchange can be done either in
column (for example; Zeba Spin Desalting Columns, 7K MWCO,
Thermo Scientific) or dialysis of antibody in 5L of 1xPBS at 4°C for few
hours to over night in dialysis cup (example: Slide-A-Lyzer MINI
Dialysis Units/Cassettes, 2,000-7,000 MWCO, Thermo Scientific).
Presence of Gelatin and BSA has significant effect on conjugation
efficiency; so both need to purify from the antibody buffer before
conjugation reaction. The supply gelatin contains18 distinct amino acids
various average mol. wts, but most likely below ~50 KDa and BSA ~66
KDa. BSA and gelatin completely can remove from antibody solutions
using MelonTM Gel (Thermo Scientific, Supplementary 1) or Using
Amicon Ultra 0.5 mL Centrifugal Filter Unit with Ultracel 100K
membrane (Millipore) both Gelatin and BSA can be washout a great
amount from the antibody containing solution.
B. Concentration of antibody after dialysis or purification on Melon
gel: Using Amicon Ultra 0.5mL Centrifugal Filter Unit with Ultracel 10K
membrane and follow manufacture instructions. Measure concentrated
antibody concentration using Nano drop or Bradford protein assay.
C. Antibody Concentration: Generally, 1-4 mg/ml concentration and
amount more than 10 g give optimal results for antibody concentration
in the reaction buffer. Antibody concentration less than 1 mg/ml, they
need to be concentrated to at least 1mg/ml using Amicon Ultra 0.5mL
Centrifugal Filter Unit with Ultracel 10K. Antibody concentration recalculates using Nano drop or Bradford protein assay; usually during
concentration process antibody is losses more than 5% sometimes.
2. Oligonucleotide/DNA: Azide modified DNA (100M) in MQ
water.
3. DBCO-NHS ester (MW: 430.45) cross linker preparation: Fresh
4-10mM cross linker need to prepared in DMSO or DMF for
conjugation reaction. (Stability: DBCO-NHS ester stable at -20°C
around one year but the DBCO-NHS dissolved in DMSO can be store
for few months at -20°C for use but keep note that NHS ester are
moisture-sensitive so it can hydrolysis).
Note: DMSO is a very hygroscopic liquid and should protected from
exposure to moisture.
4. Quenching buffer: 1M Tris-Hcl, pH 8.0 at room temperature.