Fluorescent dyes are important tools for biomolecular labeling. Fluorescein derivatives are widely labeled on reagents used in fluorescence microscopy, flow cytometry and immunofluorescence detection. Usually, dyes such as biotin and FITC can be arbitrarily labeled at the N-terminal or C-terminal of polypeptide. But we recommend N-terminal markers, which have a higher success rate, take less time, and are easier to operate. Since polypeptides are synthesized from the C-terminal to the N-terminal, the N-terminal modification will be the last step in the solid-phase synthesis, and no additional coupling steps are required. On the contrary, if it is a C-end tag, additional steps are required, and the process is more complicated.
Ordinary fluorescent modification
| Fluorescent Modified Chinese Name | N-end | N-end with linker |
| Biotinylated polypeptide | Biotin- | Biotin-Ahx- |
| Fluorescein isothiocyanate | FITC- | FITC-Ahx- |
| 5-carboxyl fluorescein | 5-FAM- | 5-FAM-Ahx- |
| Dansulfonyl fluorescein | Dansyl- | Dansyl-Ahx- |
| 5-carboxyl tetramethylrhodamine | TMR- （TAMRA-） | TMR-Ahx- （TAMRA-Ahx-） |
Fluorescent Modification by Amino Linkage of Lys Side Chain
| N-terminal polypeptide | Peptide sequence intermediate | C-terminal polypeptide |
| Lys(Biotin)- | -Lys(Biotin)- | -Lys(Biotin) |
| Lys(FITC)- | -Lys(FITC)- | -Lys(FITC) |
| Lys(5-FAM)- | -Lys(5-FAM)- | -Lys(5-FAM) |
| Lys(Dansyl)- | -Lys(Dansyl)- | -Lys(Dansyl) |
| Lys(TMR)- | -Lys(TMR)- | -Lys(TMR) |
| Lys(Dnp)- | -Lys(Dnp)- | -Lys(Dnp) |
We usually do the excitation wavelength and emission wavelength of fluorescent substances
| Fluorescent group | Ex（nm） | Em（nm） | Fluorescent group | Ex（nm） | Em（nm） |
| Hydroxycoumarin | 325 | 386 | R-phycoerythrin (PE) (489) | 565 | 578 |
| Danesulfonyl chloride | 340 | 578 | Rhodamine Red-X | 560 | 580 |
| AMC | 345 | 445 | Tamara | 565 | 580 |
| Methoxycoumarin | 360 | 410 | Alexa fluor 555 | 556 | 573 |
| Alexa fluor series | 345 | 442 | Alexa fluor 546 | 556 | 573 |
| Amino coumarin | 350 | 445 | Rox | 575 | 602 |
| Dabcyl | 453 | – | Alexa fluor 568 | 578 | 603 |
| Cy2 | 490 | 510 | Texas Red | 589 | 615 |
| FAM | 495 | 517 | Alexa fluor 594 | 590 | 617 |
| Alexa fluor 488 | 494 | 517 | Alexa fluor | 621 | 639 |
| FITC | 495 | 519 | Alexa fluor 633 | 650 | 668 |
| Alexa fluor 430 | 430 | 545 | Cy5 (625) | 650 | 670 |
| 5-FAM | 492 | 518 | Alexa fluor 660 | 663 | 690 |
| Alexa fluor 532 | 530 | 555 | Cy5.5 | 675 | 694 |
| HEX | 535 | 556 | TruRed | 490; 675 | 695 |
| 5-TAMRA | 542 | 568 | Alexa fluor 680 | 679 | 702 |
| Cy3 | 550 | 570 | Cy7 | 743 | 767 |
| TRITC | 547 | 572 | Cy3.5 | 581 | 596 |