This protocol outlines antibody conjugation by NHS-esters.
|Item||Item article number||Distributor||Vendor|
|UltraPure™ DNase/RNase-Free Distilled Water||10977035||ThermoFisher||Invitrogen|
|0.5 M NaHCO3, pH 8|
|NHS-ester or similar|
|Zeba Spin Desalting Columns, 0.5 ml,40K MWCO
BSA, azide, or glycine that are often added by the manufacturer to the antibody, this step is to remove such reagents from the antibody buffer.
Amine-containing compounds (glycine, Tris, or ammonium ions) and stabilising proteins (BSA) will interfere with the NHS to amino labelling.
1️⃣ Add 500 ul of 1X PBS to moisten the cellulose membrane of the Amicon ultra 10K 0.5mL (UFC501096).
2️⃣ Centrifuge at 4000 g for 3 min and the discard both the flow-through and the PBS at the bottom of the device.
3️⃣Wash the antibody in 1X PBS by taking 80 ul of antibody at 2 mg/mL and fill up with 500 µL of 1X PBS.
Add desired antibody and amount together with 500 µL of 1X PBS and centrifuge for 10 min @4000 g.
4️⃣ Discard the flow-through and add 500 µL of 1X PBS and centrifuge again for 10 min @4000 g.
5️⃣ Collect purified antibody from the device by reverse spin at 1000 g for 2 min.
6️⃣Measure the absorbance of the antibody with a NanoDrop at 280 nm (A280). Write down the concentration (mg/mL) of the antibody.
1️⃣Take out the NHS ester from the desiccator in the fridge and place within
Dissolve amine-reactive compound (NHS ester) in DMSO to 10 mg/mL (i.e. 100 µL of DMSO is added to 1 mg compound).
1️⃣Calculate the amount of NHS ester in DMSO you need to add to the antibody.