Conjunction of DNA and primary antibody

Dear Noorsher Ahmed,

The following is the detailed protocal for the conjunction of DNA and primary antibody:

1.100K MWCO Amicon Ultra 0.5 mL filter was firstly rinsed with reaction buffer, and centrifuged at 14,000 g for 10 min.
2.After removing the reaction buffer, the filter can be used to purify commercial primary antibodies (PA).
3.PA was mixed with reaction buffer to a volume of 0.5 mL, and then centrifuged at 14,000 g for 10 min.
4.Then, filtrate was removed and the PA was recovered to 0.5 mL with reaction buffer.
5.Repeat step 3-4 at least three times. After the last centrifugation, PA was collected.
6.The collected PA was quantified with nanodrop and suspended in reaction buffer [55 mM sodium phosphate, 150 mM sodium chloride, and 20 mM EDTA (pH 7.2)] to make 1 mg/mL solutions, followed by a 2-hour incubation of 80-fold excess of sulfo-SMCC (dissolved in anhydrous DMSO) at RT to activate the amino group of primary antibodies.
7.A 10K MWCO Zeba spin desalting column was used to separate out redundant sulfo-SMCC by centrifuge ultrafiltration for three rounds.
8.In parallel, we reduced the thiol-modified initiators with DTT at a molar ratio of 1:500 in the reaction buffer for 1 hour at 37°C. Excess DTT in the thiolated DNA solution was removed by a 3K MWCO Zeba spin desalting column. 
9.The sulfo-SMCC–activated primary antibodies were mixed with a threefold molar excess of reduced initiators and reacted at RT for 2 hours. 
10.Lastly, protein-DNA conjugates were further purified by the 10K MWCO Zeba Spin Desalting Column and stored at 4°C for short-term usage or at −20°C until used.

As an alternative, a strategy based on the NHS-PEG4-N3 cross-linking agent is another option (Protein spherical nucleic acids, Chad A. Mirkin).

Best wishes!