DyLight

Add a quantity of the DyLight 649 dye to the dendrimer solution to provide at least a 1.25-fold molar excess of dye over the amount of dendrimer present (for nonaqueous reactions) or a 6-15-fold molar excess for aqueous reactions. Mix well to dissolve. The optimal amount of dye added should be determined experimentally by preparing a series of conjugates using different molar ratios of dye-to-dendrimer. 

RPE-Alexa 610, or RPE-DyLight 594 RPE-Alexa 647 or RPE-DyLight 594 RPE-DyLight 649 DyLight 649,... 

The following protocol is based on the methods recommended by Thermo Fisher for use of the cyanine dye DyLight 649. Antibody reduction is based on the methods of Sun et al. (2005), which results in partially reduced bispecific immunoglobulin containing available thiols in the hinge region for labeling. 

Manufacturers of Alexa (Invitrogen), Dylight (Thermo Fisher Scientific), and ATTO (ATTO-Tec, also available from Sigma-Aldrich) dyes offer a broad choice of fluorophores covering the entire visible spectrum in small increments. Red dyes from these lines are reported to be significantly... 

Full in-house oligonucleotide synthesis with 5 end incorporation of dye phos-phoramidites. Due to wide availabifity of commercially synthesized oligonucleotides, full synthesis of RNA oligos in individual labs is not frequendy carried out these days and choices of commercially available dye phosphoramidites are limited. Thermo Fisher Scientific (www. thermo.com) provides a selection of DyLight phosphoramidites that have spectral properties analogous to Cy3, Cy5, and Cy5.5 dyes. 

DyLighP 547 and DylighU 647 Monoclonal Antibody Labeling Kits (Cat. Nos. 53009 and 53015, Pierce Biotechnology) containing DyLight 547 and 647 NHS Esters, dimethylformamide (DMF), Borate buffer (0.67 M), and Zebra Desalt Spin Columns. 

Complete steps 1-10 rapidly without interruption. Once the DyLight dyes are reconstituted, they must be used immediately (see Note 3). 

Add 5 pi of DyLight 647 to each of the corresponding tubes from step 2. Vortex the four microfuge tubes gently and briefly centrifuge the microfuge tubes to collect the samples at the bottom. 

Place each column into a fresh 2-ml collection tube and label two tubes for each sample A-DyLight 547, A-DyLight 647, B-DyLight 547, B-DyLighP 647 (8-column/tube assemblies in total). 

Transfer the entire sample of IgG A-DyLighf M 547 and IgG B-DyLight 647 (200 pg total from Subheading 3.3) to the Mix 1 tube from step 1. 

Save the DyLight 547 and 647 images as separate TIFF files. The single-file TIFF format is the most useful for quantifying data from the images. Other file formats, such as Bitmap (BMP), may be appropriate for other tasks (see Note 8). 

Fig. 3. An illustration of results from the two-slide dye-swap. In Mix 1, control IgG is labeled with DyLight 647 dye (red) and ovarian cancer patient IgG is labeled with DyLight M 547 dye (green). In Mix 2, the dyes are swapped, so control IgG fluoresces green, whereas ovarian cancer IgG fluoresces red. A portion of each array slide is enlarged to clearly illustrate that green spots denoting ovarian cancer autoantibody reactivity in Mix 1, fluoresce red in Mix 2.

Buffer used in Subheading 3.1 is compatible with all of the DyLight labeling reagents used in Subheading 3.2. 

The Zebra Desalt Spin Columns that come with the DyLight Labeling Kits remove free dye. These columns contain a desalting resin and molecular weight cutoff. They perform well in desalting small sample volumes, providing excellent protein recovery and > 95% retention of small molecules and salts (<7 kDa). 

Immunofluorescence species-specific secondary antibodies (1 500) conjugated with fluorophores at different excitation wavelengths depending on the microscope (e.g., 488 nm for green or 594 nm for red fluorescence). Fluorochrome-labeled secondary antibodies can be obtained from different suppliers, e.g., Alexa Fluor, Invitrogen or DyLight Fluor, Thermo Fisher Scientific, Waltham, MA (caution light sensitive, store at4°C). 

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