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Mix-n-Stain™ Small Ligand Labeling Kits

The world’s simplest labeling protocol: Mix and then Stain ~ it really is that simple!

Mix-n-Stain™ CF™ dye small ligand labeling kits are designed for rapid labeling of small (MW ~ 150 – 5,000) and relatively high affinity biological ligands (or substrates). Labeling takes about 30 minutes, without a final purification step. The ligands to be labeled must contain an aliphatic amine group that is not required for biological activity of the ligand. The amine group will form a covalent linkage with the reactive CF dye provided in the kit. For example, suitable ligands or substrates include SNAP-tag®, CLIP-tag™ and HaloTag® ligands with an aliphatic amine. Many other small ligands are also possible candidates if they meet the criteria described above.

Simply mix your small molecule ligand with the reaction buffer and the optimally formulated dye provided, and incubate for 30 minutes, followed by a brief 5 minute quenching step. No reactive Mix-n-Stain™ dye is available at the end of labeling; therefore the conjugate is ready for cell staining and imaging without further purification. Mix-n-Stain™ labeled ligands perform comparably to commercially-available pre-labeled ligands (Figure 1), at a fraction of the cost.

A major advantage of Mix-n-Stain™ kits is our superior CF™ dyes, many of which are brighter and more photostable than Alexa Fluor® dyes and other similar commercial dyes. Several of our CF™ dyes, such as CF™405M, CF™633, CF™640R, CF™647, and CF™680R, have been demonstrated to be ideal probes for super-resolution and single molecule imaging (see references here). With a choice of ten CF™ dyes for cell surface labeling and three CF™ dyes for intracellular labeling, you have significant freedom to label your small molecule ligand with colors ranging from blue to near-infrared, making multi-color detection simple and affordable (Figures 2, 3). See the product list at the bottom of this page for more information.

For live cell imaging, the Mix-n-Stain™ kits are ideal to generate fluorescent chemical tags that provide smaller, brighter, and more versatile alternatives to fluorescent proteins. Labeling of various chemical tags with Mix-n-Stain™ kits yielded highly efficient and specific labeling of the target proteins both on surface and inside of living cells. The Mix-n-Stain kits have also been proven compatible with fixed cell imaging (Figure 3) and gel-based assays (see the Mix-n-Stain™ Small Ligand Flyer for more details).

 

CF488A CLIP live cell imaging
Figure 1. Live cell imaging of HeLa cells expressing CLIP-NK1R labeled with (A) CLIPamine conjugated to green fluorescent CF488A using Mix-n-Stain compared to (B) CLIP-surface 488 from New England Biolabs (NEB catalog no. S9232S). Cell nuclei were stained blue with Hoechst 33342 (catalog no. 40046).

 

Three color live cell
Figure 2. Three color imaging in live HeLa cells. Cell surface protein ADRβ2 was labeled using the CF488A Mix-n-Stain kit via the SNAP-tag (green). Mitochondrial protein Cox8A was labeled using the CF540 Mix-n-Stain kit via the CLIP-tag (red). Cell nuclei were stained blue with Hoechst 33342 (cat. no. 40046).
Three color fixed cell
Figure 3. Three color imaging in fixed HeLa cells. Cell surface protein NK1R was stained with CLIP-tag labeled with the CF488A Mix-n-Stain kit (green). Cells were fixed, permeabilized, stained with CF633 phalloidin (catalog no. 00046, red), and mounted with EverBrite Mounting Medium with DAPI (catalog no. 23002; nuclei: blue).

Mix-n-Stain™ small ligand labeling kits for cell surface labeling are available in your choice of 10 CF™ dye colors for labeling ligands for cell surface targets, and three CF™ dye colors for ligands that bind intracellular targets. Also see our Mix-n-Stain™ Antibody Labeling Kits and other Reactive Dyes and Biotin Reagents.

DyeCatalog no.Spectrally similar dyesEx (nm)Em (nm)Staining
CF™405M92362Pacific Blue™, BD Horizon™ V450408452Surface
CF™488A92350FITC, Alexa Fluor® 488490515Surface
CF™56892351Rhodamine Red, Alexa Fluor® 568562583Surface
CF™59492352Texas Red®, Alexa Fluor® 594593614Surface
CF™63392353Cy®5, Alexa Fluor® 633630650Surface
CF™640R92354Cy®5, Alexa Fluor® 647642662Surface
CF™64792359Cy®5, Alexa Fluor® 647650665Surface
CF™660C92360Alexa Fluor® 660667685Surface
CF™68092361Cy®5.5, Alexa Fluor® 660, IRDye®680LT681698Surface
CF™680R92355Cy®5.5, Alexa Fluor® 660, IRDye®680LT680701Surface
CF™40892356Pacific Blue™, BD Horizon™ V450408450Intracellular
CF™50092357FITC, Alexa Fluor® 488500510Intracellular
CF™54092358Tetramethylrhodamine (TAMRA), TRITC, Alexa Fluor® 546, Cy®3540570Intracellular
CF™55592364Alexa Fluor® 555, Cy®3, TRITC555585Intracellular
CF™65092363Cy®5, Alexa Fluor® 647650670Intracellular

Mix-n-Stain and CF dyes are covered by US and international patents, and are trademarks of Biotium, Inc. SNAP-tag and CLIP-tag are trademarks or registered trademarks of New England BioLabs; HaloTag is a registered trademark of Promega Corporation; Pacific Blue, Texas Red, and Alexa Fluor are trademarks or registered trademarks of Invitrogen Corp; BD Horizon is a trademark of BD Biosciences; Cy Dye is a registered trademark of GE Healthcare; IRDye is a registered trademark of LI-COR Biosciences.