Mix-n-Stain™ CF® Dye Small Ligand Labeling Kits

For CF® dye, cyanine dye, FITC, biotin, DNP, and dig kits, see the Mix-n-Stain™ Kit Compatibility and Protocol Selection Flowchart, below. We also recommend downloading the updated Product Protocol and completing the pre-labeling checklist to find the right protocol and kit size to use for each antibody you wish to label. Learn more about CF® dyes on our CF® Dye Technology Page, or download the CF® Dye Brochure.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

There is no need to measure the dye amount or vary the reaction time as long as the amount of your antibody to be labeled falls within the range specified for each kit. With Mix-n-Stain™ labeling kits optimal labeling is ensured because of the proprietary dyes and reaction buffer.

The kits are optimized for labeling antibodies with a concentration between 0.5-1.0 mg/mL. If your antibody solution is too dilute, you can concentrate it by centrifugation using the ultra-filtration vial provided in the kit. If your antibody solution is too concentrated, you can dilute it with 1x PBS. Antibody concentrations outside the recommended range may result in either under or over labeling.

The exact chemical structures of CF® dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF® dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF® dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF® dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF® dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF® dye invention that makes CF® dyes superior to other commercial dyes.

Rhodamine-based CF® dyes (designated R) generally have better photostability but weaker fluorescence than their cyanine-based equivalents (designated C). Therefore, rhodamine-based near-IR CF® dyes are a better choice for microscopy, while cyanine-based CF® dyes are more ideal for flow cytometry, Western blotting, and other applications where photobleaching is less of a concern. Another factor to consider is the size of the dyes. Some of the cyanine-based near-IR CF® dyes are much larger than the rhodamine-based equivalents. For antibody labeling, either version of the CF® dyes is suitable. However, for applications where the dye size may cause a steric problem, the smaller dye may be a better choice.

All three of these dyes can be excited by the 405 nm laser (or UV mercury lamp). They differ in their emission wavelengths. CF®405S has blue fluorescence emission at 431 nm, similar to AlexaFluor® 405, Cascade Blue®, and DyLight™ 405 (Thermo Scientific). CF®405M has longer wavelength blue fluorescence emission at 452 nm, similar to Horizon™ V450 (BD Biosciences) and Pacific Blue® (Thermo Scientific). CF®405L has orange fluorescence emission at 545 nm, similar to Pacific Orange® (Thermo Scientific). We recommend choosing the dye that best fits your instrument’s detection settings. See our CF® Dye Quick Reference Table and CF® Dye Selection Guide for more information on CF® dyes.