Hahn Lab Dye Kit. Contents of dye kit:

Transcription

1 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: , ) 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: , ) Toutchkine, A., Dan-Vinh Nguyen, D., and Hahn, K.M. Merocyanine dyes with improved photostability. Org. Lett. 9: , ) 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: , ) Toutchkine, A., V. Kraynov, and K. M. Hahn. Solvent-Sensitive Dyes to Report Protein Conformational Changes in Living Cells, J. Amer. Chem. Soc., 125: , ) Bark, S. J., and K.M. Hahn. Fluorescent indicators of peptide cleavage inside living cells. Methods, 20: ,

2 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 hahnlab.com 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 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 µL 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 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

3 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 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,

4 Mero53 code: si-so-ia mw: DMSO MeOH BuOH Water Mero53 on protein mimic Water MeOH BuOH DMF

5 Mero58 code: TD-BA-sIA mw: DMF MeOH BuOH Water Mero58 DMSO Dioxane MeOH BuOH

6 Mero59 code: TD-SO-sIA mw: DMF MeOH BuOH Water Mero59 DMSO Dioxane MeOH BuOH

7 Mero60 code: I-Pht-sIA mw: DMSO MeOH BuOH Water < DMF Mero60 on protein mimic Water < MeOH BuOH DMF DMSO Mero60 DMSO Dioxane MeOH BuOH

8 Mero61 code: I-BA-sIA mw: DMSO MeOH BuOH Water DMF Mero61 on protein mimic Water MeOH BuOH DMF DMSO Mero61 DMSO Dioxane MeOH BuOH

9 Mero62 code: I-TBA-sIA mw: DMSO MeOH BuOH Water DMF Mero62 on protein mimic Water MeOH BuOH DMF DMSO DMSO Mero62 Dioxane MeOH BuOH DMF* Octanol*

10 Mero76 code: I-BA-sIA mw: DMSO MeOH H 2 O Mero61 DMSO Dioxane MeOH BuOH

11 Mero87 code: I-SO-sIA mw: DMSO MeOH BuOH Water Mero87 on protein mimic Water MeOH BuOH DMF DMSO DMSO Mero87 Dioxane MeOH BuOH DMF* Octanol*

12 Mero166 code: I(2AM)- BA-N3 mw: DMSO MeOH OcOH :1 H 2 O/MeOH Mero61 DMSO Dioxane MeOH BuOH

13 Mero199 Dye that undergoes ratiometric change in excitation, and contains photostability enhancements. mw: solvent Exc max Em max QY x QY Water/MeOH MeOH BuOH mw: solvent Exc max Em max QY x QY H2O/MeOH MeOH BuOH CH2Cl