Goat Anti-tdTomato DyLight antibodies are essential for detecting the tdTomato red fluorescent protein in molecular biology, cell labeling, microscopy, and flow cytometry workflows. These antibodies are commonly conjugated with DyLight dyes to improve signal brightness, reduce background noise, and allow multi-channel detection in experiments involving fluorescent protein reporters.

This detailed overview covers the biochemical properties, imaging applications, conjugation strategies, and data acquisition techniques involving Goat Anti-tdTomato DyLight, with direct references to authoritative sources from .edu and .gov domains.

tdTomato Protein: Spectral Features and Expression Use

tdTomato is a tandem dimer red fluorescent protein derived from DsRed, optimized for higher brightness and lower cytotoxicity. It has an excitation maximum at 554 nm and emission at 581 nm, making it compatible with 561 nm laser systems used in flow cytometry and confocal microscopy.

Resources describing tdTomato spectral properties include:

• Stanford Bio-X Imaging Core

• NIH Fluorescent Protein Database

• Addgene tdTomato overview

tdTomato is commonly used as a reporter gene in genetically modified organisms and cellular systems. Examples include:

• Transgenic zebrafish models from UCSD

• Neuronal tracing studies at Janelia Research Campus

Antibody Generation and Validation

Polyclonal goat antibodies targeting tdTomato are produced via peptide immunization, followed by affinity purification. The purified antibodies show:

• High specificity for tdTomato and tdTomato-tagged constructs

• Low cross-reactivity with mCherry, DsRed, and other RFPs

• Compatibility with both fixed and native samples

Batch validation studies are often published or referenced by:

• Johns Hopkins School of Medicine Core Services

• Rockefeller University Proteomics Core

DyLight Fluorophore Conjugation

The DyLight series of dyes offers superior performance in brightness, pH stability, and photostability. Common conjugates include:

Spectral matching tools are available at:

• NIH Spectrum Viewer

• Biolegend Fluorophore Selector

Immunocytochemistry and Imaging Applications

When applied in immunocytochemistry (ICC), Goat Anti-tdTomato DyLight antibodies enable the enhanced detection of tdTomato beyond the native signal. This is particularly useful in:

• Low-expression reporter systems

• Tissues with high autofluorescence

• Samples requiring multiplexed imaging

Detailed protocols are available from:

• UCSF Nikon Imaging Center

• University of Michigan BRCF

• Harvard Neurobiology Imaging Core

Common workflow:

1. Fix cells/tissue with 4% paraformaldehyde

2. Permeabilize using Triton X-100

3. Block with 5% normal serum

4. Incubate with Goat Anti-tdTomato DyLight

5. Wash and mount with DAPI-containing medium

This procedure is compatible with downstream processing by:

• Zeiss LSM series confocal systems

• Olympus FV and FLUOVIEW systems

Flow Cytometry Applications

tdTomato detection by flow cytometry using DyLight-conjugated goat antibodies allows for:

• Signal amplification of weak tdTomato reporters

• Precise gating of tdTomato-expressing cell populations

• Panel expansion using multiple fluorophores

Relevant instrumentation includes:

• BD FACSCanto and LSR II with 561 nm lasers

• Beckman Coulter CytoFLEX

For control setup and compensation, consult:

• NIH Center for Cancer Research Cytometry Core

• UCSF Flow Core Guidelines

Western Blotting Utility

Unlike unconjugated antibodies, Goat Anti-tdTomato DyLight conjugates can be used directly in Western blotting, eliminating the need for secondary antibodies. This minimizes:

• Background signal

• Antibody incubation steps

• Time required for blot development

Protocols are documented by:

• NIH NINDS Antibody Resource

• UC Davis Proteomics Core

Tissue and Organism Compatibility

Validated in:

• Murine tissue (brain, heart, lung)

• Zebrafish and Drosophila models

• Human iPSC-derived cells

• Fixed organoids and spheroids

Reference workflows:

• Yale Stem Cell Core

• University of Washington Cell Imaging

Storage, Handling, and Reagent Safety

• Store between 2–8°C, protected from light

• Do not freeze unless indicated

• Avoid multiple freeze-thaw cycles

Refer to:

• NIH Lab Safety Plan

• CDC Laboratory Biosafety Manual

Common Troubleshooting Notes

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Keywords are compiled based on:

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Further Learning and External Resources

• NIH Fluorescent Antibody Protocols

• UCLA Flow Cytometry Support

• University of Iowa Core Facilities

• FDA RUO Antibody Guidelines

• CDC Chemical and Reagent Handling Guide