Supplementary 1:
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. For standard conjugation reaction need to avoid any kinds
of additives in buffer for the conjugation reaction. So antibody need to be only
in PBA with 1xPBS buffer exchange either in column (for example; Zeba Spin
Desalting Columns, 7K MWCO, Thermo Scientific) or dialysis the antibody in
5L of 1xPBS at 4°C for few hours to over night in dialysis cup (example: SlideA-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 Melon TM 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 (280nm for antibody and 260nm Oligo) 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 re-calculates using Nano drop or
Bradford protein assay; usually during concentration process antibody is losses
more than 5% sometimes.
2. Oligonucleotide/DNA: Aldehyde modified DNA (100M) in MQ water.
3. Light sensitive Sulfo-SANH (290.2 MW) cross linker preparation: Fresh
20mM cross linker need to prepared in DMSO/DMF for conjugation reaction.
Calculation: 20mM Sulfo-SANH for 1mg of Sulfo-SANH dissolved in 172.25ul
of DMSO. Note: DMSO is a very hygroscopic liquid and should protected from
exposure to moisture.
4. Aniline Preparation: 10mM aniline need to be freshly prepared in
1xconjugation buffer (100mM NaHPO4, 150 mM NaCl, pH 6.0). Note: **
aniline is difficult to dissolve so prepare at least 30 min before needed and heat
treat.