Dylight 549

Manufactured by Rockland Immunochemicals

Sourced in United States

DyLight 549 is a fluorescent dye that can be used for labeling proteins, antibodies, and other biomolecules. It has an absorption maximum at 562 nm and an emission maximum at 576 nm, making it suitable for detection in the yellow-orange region of the visible spectrum.

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5 protocols using dylight 549

Immunofluorescence and Immunoblotting Analysis

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Immunofluorescence assays were performed as described previously [9 (link)]. Primary antibodies against p-H2A.X (Ser139), and p-53BP1 (Ser1778) (Cell Signaling Technology), were diluted 1:250 with PBS with 0.1% Tween-20 (PBST), and incubated overnight at 4° C. Secondary antibodies were either Alexa Fluor 594-conjugated secondary (Invitrogen), DyLight^TM 488 (Rockland) or DyLight™ 549, diluted 1:1000 in PBST. Slides were mounted with Prolong Gold (Invitrogen). Cellular lysates were produced and analyzed by immunoblotting as described previously [9 (link)]. Primary antibodies were against p-H2A.X (Ser139), H2A.X, p-53BP1 (Ser1778), 53BP1, CHK1 (Cell Signaling Technology) p-CHK1 (Ser345, R&D Systems) and GAPDH (Santa Cruz). Results were quantified using ImageJ and Image Lab from BioRad.

Colavito S.A., Platt J.T., Held M.A., Liu Z., Sokup R, & Stern D.F. (2019). Combinatorial drug screening of mammary cells with induced mesenchymal transformation to identify drug combinations for triple-negative breast cancer. Oncotarget, 10(47), 4822-4839.

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Detecting Rotavirus: Antibody-Based Assays

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To detect RV, we used rabbit anti-RV strain Alabama^{33 (link)} (IF, 1:80,000; western blot, 1:3000), guinea pig anti-NSP2^{34 (link)} (IF, 1:5000), and rabbit anti-NSP4 aa120-147^{35 (link)} (western blot, 1:3000). Secondary antibodies for IF were donkey anti-rabbit AlexaFluor 568 (Invitrogen) and donkey anti-guinea pig Dylight 549 (Rockland), both at 1:2000. For western blots, we used mouse anti-GAPDH monoclonal antibody (Lifetein) (1:5000) and secondary antibodies alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Southern Biotech) (1:2000).

Chang-Graham A.L., Perry J.L., Strtak A.C., Ramachandran N.K., Criglar J.M., Philip A.A., Patton J.T., Estes M.K, & Hyser J.M. (2019). Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum. Scientific Reports, 9, 10822.

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Immunohistochemical Analysis of Cx43 in Infected Mouse Hearts

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Heart tissues from the infected A129 mice and controls were collected at 8 dpi and fixed in 4% paraformaldehyde (PFA) at 4°C overnight, dehydrated with 30% sucrose in PBS, and frozen in optimal cutting temperature (OCT) compound (Leica, UK) for cryosections. The sections of hearts (15 μm thick) were washed three times in PBS, blocked by 2% bovine serum albumin (BSA), and immunostained by anti-Cx43 antibody (no. C6219; Sigma, USA). After rinsing with PBS, the primary antibody was detected by goat antirabbit antibodies conjugated with DyLight 549 (no. 611-142-002; Rockland, USA). Antibodies were diluted in blocking buffer. Finally, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (no. D1306; Invitrogen, USA) for 5 min. Images of sections were taken under an Axio Imager Z2 microscope (Zeiss, Germany).

Li S., Armstrong N., Zhao H., Cruz-cosme R., Yang H., Zhong C., Fu W., Wang W., Yang D., Xia N., Cheng T, & Tang Q. (2022). Zika Virus Infection Downregulates Connexin 43, Disrupts the Cardiomyocyte Gap Junctions and Induces Heart Diseases in A129 Mice. Journal of Virology, 96(21), e01373-22.

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Immunofluorescence Microscopy of HA-tagged Proteins

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Immunofluorescence microscopy was carried out as described previously [15] (link). Anti-HA tag antibody (1:200, 11867423001; Roche Applied Science) and anti-acetylated α-tubulin antibody (1:200, ab24610; abcam) were used as primary antibodies. Anti-mouse IgG antibody conjugated with Alexa Fluor 350 (1:200, A11045; Life technologies) and anti-rat IgG antibody conjugated with DyLight 549 (1:200, 612-142-120; ROCKLAND) were used as secondary antibodies. Images were taken with a CCD camera (DP73; Olympus).

Ide T., Mochiji S., Ueki N., Yamaguchi K., Shigenobu S., Hirono M, & Wakabayashi K.I. (2016). Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii. Biochemistry and Biophysics Reports, 7, 379-385.

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Fluorescent Labeling of Cellular Organelles

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HeLa cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, #08458-45) containing 10% fetal bovine serum (Equitech Bio, #268-1). Rabbit polyclonal anti-TOM20 antibody was purchased from Santa Cruz Biotechnology (#sc-11415), and mouse monoclonal anti-GM130 antibody was from BD Biosciences (#610822). Alexa Fluor 555 conjugated anti-mouse IgG (#A31570), Alexa Fluor 555 conjugated anti-rabbit IgG (#A31572), Alexa Fluor 555 conjugated streptavidin (#S21381), Alexa Fluor 647 conjugated streptavidin (#S21374), and Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (#W32466) were from Thermo Fisher Scientific. Biotinylated WGA (#B-1025) was from Vector Laboratories. DyLight 549 (#S000-42) and DyLight 649 (#S000-43) conjugated streptavidins were from Rockland. HiLyte Fluor 555 (#AS-60666) and HiLyte Fluor 647 (#AS-60667) conjugated streptavidins were from Anaspec. iFluor 555 (#16989) and iFluor 647 (#16996) conjugated streptavidins were from AAT Bioquest. ATTO 647 (#AD647-61) streptavidin was from ATTO-TEC. SPICA dye-conjugated streptavidin was from Fujifilm Wako Chemicals. Biotin-SP AffiniPure goat anti-rabbit IgG (#111-065-144) was purchased from Jackson ImmunoResearch.

Tanida I., Yamaguchi J., Suzuki C., Kakuta S, & Uchiyama Y. (2023). Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells. Heliyon, 9(6), e17394.

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