Labelling Strategies

Tutorial: Labeling Strategies for Fida Experiments

Introduction

This tutorial provides an overview of various labeling strategies for Fida experiments, focusing on fluorescent detection-based assays. It covers selecting labels, detectors, and labeling approaches suitable for different experimental setups. Watch the video.

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Label Selection and Detectors

• Fida Assay Basics: Fida assays rely on fluorescent detection, requiring careful consideration of excitation and emission wavelengths.
• Detectors: Three detectors are available, each suited to different wavelengths. Switching between detectors is a simple five-minute procedure.
• 275 nm Excitation: Allows for intrinsic tryptophan excitation, enabling label-free assays. Emission filter cuts off light below 300 nm.
• 480 nm Excitation: Ideal for blue light-absorbing molecules, emitting green, yellow, or red light. Commonly used for biological fluorescent tags and dyes.
• 640 nm Excitation: Offers high sensitivity to red light fluorescent dyes, minimizing background interference.

Labeling Strategies

Fusion Constructs with Fluorescent Proteins:

1. Suitable for assays in lysates.
2. Proteins are expressed fused to fluorescent tags (commonly green or yellow) at N or C terminals.
3. No purification needed; can be used directly in cell lysate.
4. Main limitation: size increase due to the 28 kDa fluorescent protein tags.

SNAP Tags:

1. A smaller alternative (19 kDa) to fluorescent protein tags.
2. Allows precise label positioning and access to a broad range of dyes.
3. Requires a chemical reaction for dye conjugation.‍

Covalent Labeling Kits:

1. Fida Bio offers two kits (ALC 480 and Fl 480) for labeling primary amines.
2. Suitable for all proteins, but proteins must be purified and free of primary amines.
3. ALC 480: Insensitive to pH and environmental changes.
4. Fl 480: Sensitive to pH and environmental changes, suitable for small molecule binding studies.

Thiol Labeling:

1. Offers control over label placement.
2. Requires at least one free cysteine and avoidance of reducing agents.

Click Chemistry:

1. Involves incorporating unnatural amino acids with an alkyne group.
2. Requires specialized expression systems and DNA sequences.
3. Offers complete control over label placement.

Labeled Peptides or Nucleotides:

1. Recommended to label during synthesis.
2. Suitable for assays with minimal consumption and high sensitivity.

Noncovalent Labeling Strategies:

1. Commonly use Oregon Green and Tris NTA nickel complex for histidine-tagged proteins.
2. Proteins remain native, but affinity constants must be considered.
3. High specificity options include labeled nanobodies or antibodies.

Biomolecule Specific Dyes:

1. Examples: Thioflavin T for amyloid fibrils, SYBR gold for nucleotides.
2. Fida assays benefit from no size bias in these dyes.
3. Generally require higher concentrations due to lower affinity.