Anti-GFP nanobody Alexa594 conjugation

Materials

1) TCEP HCl, Cat#75259, Sigma-Aldrich

2) P6 spin-column (Cat# 7326227, BioRad)

3) Phosphate Buffered Saline without calcium chloride and magnesium chloride, Cat#D8537, Sigma-Aldrich)

4) Alexa 594 maleimide (Cat#A10256, Invitrogen)

5) Superdex 200

Proceedure

1. Start with an eppendorf tube containing a solution of nanobody (100 micrograms/0.1 mL), add 1.5 microL of tris-(2-carboxyethyl)phosphine (1 M TCEP HCl, Cat#75259, Sigma-Aldrich). The final concentration of TCEP is 15 mM.
2. Shake the mixture on ice for 10 min.
3. Pre-wash P6 spin-column (Cat# 7326227, BioRad) 3-times with labeling buffer (Dulbecco’s Phosphate Buffered Saline without calcium chloride and magnesium chloride, Cat#D8537, Sigma-Aldrich) using swinging bucket centrifuge with a centrifugal force of 1,000 x g and temperature at 4 C.
4. Transfer the mixture to the pre-washed P6 spin-column and centrifuge. Collect the solution into a new eppendorf tube.
5. Add 2 microL solution of Alexa 594 maleimide (Cat#A10256, Invitrogen) in dimethylsulfoxide (5 microgram/microL) to the solution in 4.
6. Shake the reaction mixture on ice for 1 hour.
7. Transfer the reaction mixture to a column of Superdex 200 in phosphate buffer, pH 7.4.
8. Purified fractions are eluted from the column using phosphate buffer and a peristaltic pump at 1 ml/min. The product can be visibly identified and eluted as a red band on the column. The free dye generally remains at the top of the column and is slowly eluted later. Purification is done at 4C in cold room.
9. Determine the degree of labeling by measuring OD at 280 and 594 nm.