Labeling of gp45e212c

Purpose: to label the mutagenized cysteine in gp45e212c with tags for utilization in visualizing the protein on the microscope

Contents

1. Protocol
2. Quantifying Labeling Efficiency

1. Protocol

General protocols are available at these links:

• Biotin – http://www.piercenet.com/instructions/2160748.pdf (dead link)
• Alexa Fluor 488 – <http://tools.lifetechnologies.com/content/sfs/manuals/mp00003.pdf PDF>

Alexa488, expectedly, is very light sensitive and should be treated with care (all solutions containing the reagent wrapped in foil, etc.)

My purification for gp45e212c gave me a very high protein concentration (150 uM), so I first dilute the protein down 2X using a 20 mM Tris, pH 7.0 (it is VERY IMPORTANT that the buffer is at pH 7.0 for this SMCC reaction to occur); 200 mM NaCl; 5 mM MgCl2

Labeling reagents (biotin-maleimide is in the 4C fridge and Alexa488-maleimide is in the -20C freezer) need to be at a 20X excess in the reaction

These subsequent volumes are for a protein solution of 75 uM -

1. Add 190 uL DMSO to 2 mg biotin.
2. Dab a pipet tip into the tube of solid of Alexa488-maleimide and dip into 15 uL of DMSO.
3. Add 15 uL of Biotin reagent to 200 uL 75 uM gp45e212c; similarly, add 15 uL of Alexa488 reagent to 200 uL 75 uM gp45e212c.
4. Incubate overnight rotating in the cold room.
5. After incubation, spin in P-6 gel filtration tubes to get rid of excess reagent; then, dialyze overnight in 20 mM Tris, pH 7.0; 200 mM NaCl; 5 mM MgCl2; and 10% glycerol.
6. Aliquot out and freeze.

2. Quantifying Labeling Efficiency

To assess efficiency of labeling, use a Biotin-Streptavidin upshift assay for the biotinylated gp45e212c.

For Alexa488-labeled gp45, use the extinction coefficients of Alexa488 (73000 cm-1M-1) and gp45e212c (19940 cm-1M-1) as well as Beer’s law to find concentrations (measure A493 for Alexa488 and A280 for protein on the nanodrop).