Introduction
Labeling antibodies with STORM dyes Secondary antibodies are labeled with a mixture of two amine-reactive dyes: an activator dye (Alexa Fluor 405, Cy2 or Cy3) and a reporter dye (Cy5, Alexa Fluor 647 or Cy5.5). After labeling, the number of dyes per antibody molecule should be characterized by measuring the absorption spectrum. Generally, 2 ~ 4 activator dyes and 2-8 reporter dyes per antibody molecule are acceptable. Cy3B reporter and Alexa 488 reporter do not benefit from coupling with activator dyes.
Protocol
- Mix 80 uL of dk anti-(species) IgG (1.25 mg/mL in PBS with Jackson, 2 mg/mL from abcam) with 10 uL of 1 M NaHCO3 (420mg sodium bicarbonate/5ml H20) (make fresh each time), 1.7 uL 405 and 3 uL Alexa Fluor 647 (consult most recent reaction conditions and resulting labeling ratios for that current batch of dye). React at room temperature for 10 ~ 15 min. Protect dye from water in storage, keep frozen in pure DMSO.
- During the reaction, equilibrate a Sephadex G-25 column (GE Healthcare, NAP-5) with PBS. Let fluid out of the column and then put in PBS x 3 full rinses.
- Purify the labeled antibodies on the NAP-5 column (GE healthcare):
* Add in ABs and allow to enter the column
* Add 600 uL of filter sterilized PBS, discard flow through.
* Add 300 uL of filter sterilized PBS, collect flow through in labeled eppendorf. (final volume about 250 uL)
4. Measure the degree of labeling using UV-Vis absorption. This protocol should produce about 0.3 mg/mL of labeled secondary antibody with about 2 405 and 3 Alexa Fluor 647 per protein molecule. The activities of amine reactive dyes vary from batch to batch; therefore, a second labeling reaction is usually needed to allow the amount of dye used for labeling to be adjusted accordingly. For UV-vis analysis, add 80 ul solution to be measured. Clean out the cuvette with PBS 2x before beginning, also clean 3x with PBS between each AB. Blank with PBS to begin Red dot on cuvette is down, should face the operator. Save measurements and print absorption spectra measured for lab notes.
Enter into spreadsheet to calculate AB ratios based on spectral extinction coefficients for dyes and AB and mass of antibody: