Hahn Lab Dye Kit Contents of dye kit: dye name donor/acceptor code references (see below) Mero53 si-so-ia 4 Mero58 TD-BA-sIA - Mero59 TD-SO-sIA - Mero60 I-Pht-sIA 1 Mero61 I-BA-sIA 1 Mero62 I-TBA-sIA 1 Mero76 I(2AM)-BA-COOH - Mero87 I-SO-sIA 1 Mero166 I(2AM)-BA-N3 - Mero199 AI-BA - Structures: Note that each of these dyes is discussed below with different side chain configurations. The version with iodine is the one that is used in the labeling solution. The extinction coefficient from that version should be used when determining concentrations of solutions used for labeling. Iodine quenches fluorescence but is lost upon labeling. Therefore the dye configuration without iodine, designated as on protein mimic or will most likely resemble the fluorescence of the dye once it is on the protein. For on protein mimic we have reacted the dye with a thiol-containing compound (to mimic cysteine) and then recorded the physical constants. Storage: All dyes should be stored at -20 C or colder in a well-sealed vial (i.e. seal with parafilm). Ideally this vial is kept in a sealed container containing desiccant. Warm the dye to room temperature in the dark before opening in order to prevent the uptake of moisture. Solutions in frozen DMSO lose activity over time. Dye references: 1) MacNevin, C. J.; Gremyachinskiy, D.; Hsu, C.-W.; Li, L.; Rougie, M.; Davis, T.; Hahn, K. M. Environment sensing merocyanine dyes for live cell imaging applications. Bioconjugate Chemistry. 24:215-223, 2013. 2) Toutchkine, A., Han, W.G., Ullmann, M., Liu, T., Bashford, D., Noodleman, L., and Hahn, K.M. Experimental and DFT studies: novel structural modifications greatly enhance the solvent sensitivity of live cell imaging dyes. J Phys Chem A. 111:10849-60, 2007. 3) Toutchkine, A., Dan-Vinh Nguyen, D., and Hahn, K.M. Merocyanine dyes with improved photostability. Org. Lett. 9: 2775-2777, 2007. 4) Toutchkine, A., Dan-Vinh Nguyen, D., and Hahn, K.M. Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging. Bioconjugate Chem. 18:1344-1348, 2007. 5) Toutchkine, A., V. Kraynov, and K. M. Hahn. Solvent-Sensitive Dyes to Report Protein Conformational Changes in Living Cells, J. Amer. Chem. Soc., 125:4132-4145, 2003. 6) Bark, S. J., and K.M. Hahn. Fluorescent indicators of peptide cleavage inside living cells. Methods, 20: 429-435, 2000. 1
Contact Information: Klaus Hahn Department of Pharmacology University of North Carolina at Chapel Hill CB #7365 Genetic Medicine Building 120 Mason Farm Road Chapel Hill, NC 27599-7365 hahnlab.com khahn@med.unc.edu Dye labeling protocol: 1. Dissolve a small amount of dye in 50 µl DMSO (about 0.5 mg. Do not try to weigh it static charge may cause you to lose much of the dye). If aliquots are sent pre-weighed, then a recommended dilution will be provided (skip to step 3). 2. Dilute an aliquot of the stock solution in DMSO until the absorbance max at the long wavelength dye peak (above 450 nm, at 500-700 nm for most of our dyes) is below 0.2, for accurate measurement. Determine the concentration of the dye in this solution using the dye extinction coefficient in DMSO (obtained from the dye kit information packet). This value can be used (after accounting for your dilutions) to determine the concentration of the solution produced in step 1. It is this which will be added to your protein. Note that these dyes are environment-sensing, so the extinction coefficient is solvent dependent. Do not do this procedure in some other solvent while using the DMSO extinction coefficient. 3. In the protein reaction mixture, the protein:dye ratio should be about 1:5 (for iodoacetamides). Add the concentrated solution of dye (from step 1) to the protein dropwise, with gentle mixing between drops. We try to minimize the percent DMSO in the protein solution, with < 5% being optimal. That is why we use a concentrated dye solution. The concentration of the protein is important too dilute a protein will slow reaction. In our case, we typically use a 100-300µL reaction volume (buffer: 50 mm sodium phosphate, ph 7.4) with 100 µm protein (~5mg/ml). Do not stir vigorously - bubbles etc can denature some proteins. 4. Incubate with mixing in a shaker or rotating platform at room temperature for 2 h. Note that reaction conditions (time, temperature, ph, concentrations, dye:protein ratios) can be varied to accommodate less stable proteins or to affect the number and sites of labeling. 1 For iodoacetamide or bromoacetamide dyes, use ph 6.5-7.5. The reaction vessel can be covered with foil to block light. To assist in mixing, the reaction tube may be enclosed within a larger secondary container (i.e. 50 ml conical tube) and placed on a slowly rotating stirrer. Do not rotate too quickly or you may denature some proteins - the idea is simply to keep the dye solubilized and/or in suspension. 5. Stop reaction with 5µL β-mercaptoethanol mix and incubate at RT for 5 min. This step is optional but can assist in the separation of remaining unreacted dye from the labeled protein in the subsequent purification step. 2
6. Separate unreacted dye from the labeled protein using a gel filtration medium such as Sephadex G- 15 or G-25, as appropriate for protein size. Use a narrow, long column rather than a shorter, wider column. For a 1 X 20 cm column, approximately 2 g gel is sufficient. Prepare a gel slurry using the same aqueous buffer as for the labeling reaction. Use 20x column volume over loading volume. The first-eluting colored band will be the dye-labeled protein. 7. To determine labeling efficiency, you need to determine the concentration of protein and dye, then calculate their molar ratio. Protein concentration is determined by UV absorbance, colorimetric assay, SDS-PAGE against known concentration standards, or other means. Be aware that colorimetric assays may have a readout wavelength that overlaps dye absorbance, and that the dye will affect absorbance measurements at 200-300 nm, because it has an absorbance peak there. Dye concentration is more difficult to determine, because the dye s extinction coefficient is sensitive to environment. Where we have an extinction coefficient in DMSO for the or on protein mimic, you can dissolve the protein in DMSO and then determine the dye concentration using the DMSO extinction coefficient (add a precise amount of aqueous protein solution to DMSO, keeping the amount of water in the final DMSO solution to a minimum). The DMSO is used to overwhelm the effects of the protein on the extinction coefficient. Note: Dyes should be used immediately after preparing stock solutions. Do not store the dyes as solutions even in frozen solutions at -80 degradation occurs. If possible you can use HPLC to clean up or verify the purity of dyes after prolonged storage. See us or our publications for the HPLC conditions. 1 Bark, S. J.; Hahn, K. M. (2000) Methods 20, 429-435 3
Mero53 code: si-so-ia mw: 764.67 DMSO 597 615 0.35 80470 28165 MeOH 590 608 0.14 73458 10284 BuOH 594 614 0.30 78455 23537 Water 585 608 0.03 70260 2108 Mero53 on protein mimic Water 594 618 0.04 134328 4884 MeOH 582 620 0.14 115384 16613 BuOH 590 624 0.35 119394 41566 DMF 592 624 0.46 136641 62350 4
Mero58 code: TD-BA-sIA mw: 703.55 DMF 557 578 0.14 73268 9969 MeOH 548 572 0.08 58441 4609 BuOH 556 578 0.22 64017 13925 Water 522 566 0.05 28096 1409 Mero58 DMSO 556 577 0.19 149709 28445 Dioxane 550 574 0.02 141927 2839 MeOH 542 568 0.04 128796 5152 BuOH 550 572 0.11 155438 17098 5
Mero59 code: TD-SO-sIA mw: 701.55 DMF 581 600 0.24 99929 24284 MeOH 577 598 0.05 84506 4487 BuOH 585 604 0.20 86012 17343 Water 525 588 0.02 31762 627 Mero59 DMSO 578 596 0.26 149173 38785 Dioxane 574 594 0.12 130000 15600 MeOH 572 592 0.04 111905 4476 BuOH 578 596 0.17 137755 23418 6
Mero60 code: I-Pht-sIA mw: 710.55 DMSO 593 620 0.37 126000 46620 MeOH 591 614 0.02 133000 2660 BuOH 593 616 0.16 132000 21120 Water 595 608 <0.01 106000 11 DMF 591 618 0.23 92143 21046 Mero60 on protein mimic Water 595 610 <0.01 127762 67 MeOH 591 614 0.02 127512 2892 BuOH 593 616 0.21 119061 25275 DMF 592 618 0.40 120579 48050 DMSO 595 618 0.66 124000 81870 Mero60 DMSO 590 616 0.29 201114 58323 Dioxane 570 596 0.05 120945 6047 MeOH 584 608 0.01 224426 2244 BuOH 586 613 0.10 213990 21399 7
Mero61 code: I-BA-sIA mw: 748.60 DMSO 570 595 0.37 100095 37035 MeOH 565 588 0.07 167000 11690 BuOH 570 592 0.17 146000 24820 Water 565 586 0.03 131000 3930 DMF 566 590 0.15 140147 21323 Mero61 on protein mimic Water 565 588 0.07 87169 5788 MeOH 565 588 0.13 103935 13310 BuOH 568 592 0.28 104029 28699 DMF 566 590 0.25 93750 23310 DMSO 570 594 0.55 116000 63800 Mero61 DMSO 568 590 0.40 175147 70024 Dioxane 554 576 0.02 88794 1510 MeOH 560 582 0.10 58980 5603 BuOH 564 586 0.19 81107 15410 8
Mero62 code: I-TBA-sIA mw: 764.67 DMSO 597 615 0.20 135000 27000 MeOH 590 608 0.09 140000 12600 BuOH 594 614 0.18 139000 25020 Water 585 608 0.10 36000 3600 DMF 594 612 0.24 189000 44916 Mero62 on protein mimic Water 586 608 0.06 108807 6532 MeOH 589 610 0.21 182855 38315 BuOH 594 614 0.45 196012 88215 DMF 593 614 0.42 198536 83966 DMSO 596 614 0.41 142000 58220 DMSO 590 612 0.58 239549 138938 Mero62 Dioxane 584 602 0.14 190865 26721 MeOH 584 603 0.07 234260 16398 BuOH 588 609 0.47 230097 108146 DMF* 589 611 0.94 183000 172000 Octanol* 590 609 0.99 180000 178000 9
Mero76 code: I-BA-sIA mw: 748.60 DMSO 565 594 0.18 87600 15500 MeOH 558 588 0.11 145000 15700 H 2 O 568 590 0.08 82400 6600 Mero61 DMSO 568 590 0.40 175147 70024 Dioxane 554 576 0.02 88794 1510 MeOH 560 582 0.10 58980 5603 BuOH 564 586 0.19 81107 15410 10
Mero87 code: I-SO-sIA mw: 710.55 DMSO 592 620 0.34 140000 47600 MeOH 588 620 0.06 107000 6420 BuOH 591 622 0.21 111000 23310 Water 592 616 0.02 81000 1620 Mero87 on protein mimic Water 591 616 0.02 75488 1096 MeOH 588 620 0.08 83774 6863 BuOH 592 622 0.26 100337 25554 DMF 587 618 0.42 102520 43415 DMSO 590 620 0.27 105000 28350 DMSO 586 614 0.30 170992 51298 Mero87 Dioxane 574 606 0.12 127049 15246 MeOH 582 614 0.03 161763 4853 BuOH 582 616 0.50 141426 70713 DMF* 586 615 0.97 143000 140000 Octanol* 587 617 0.98 125000 123000 11
Mero166 code: I(2AM)- BA-N3 mw: 680.66 DMSO 565 594 0.11 101800 10800 MeOH 560 586 0.05 97200 4600 OcOH 566 586 0.04 12400 5300 1:1 H 2 O/MeOH 568 590 0.09 26000 2300 Mero61 DMSO 568 590 0.40 175147 70024 Dioxane 554 576 0.02 88794 1510 MeOH 560 582 0.10 58980 5603 BuOH 564 586 0.19 81107 15410 12
Mero199 Dye that undergoes ratiometric change in excitation, and contains photostability enhancements. mw: 874.83 solvent Exc max Em max QY x QY Water/MeOH 578 607 123000 0.15 18500 MeOH 589 613 180000 0.05 9000 BuOH 600 620 190000 0.25 47500 mw: 426.47 solvent Exc max Em max QY x QY H2O/MeOH 578 607 268162 0.148 39688 MeOH 589 613 369602 0.047 17371 BuOH 600 620 284901 0.246 70086 CH2Cl2 607 625 440576 0.011 4846 13