Janelia Fluor® 525 conjugation kit

(HB9768)
Technical documents: Datasheet

Product overview

Name Janelia Fluor® 525 conjugation kit
Biological description

Overview

The Hello Bio Janelia Fluor® 525 conjugation kit allows the conjugation of antibodies and proteins to Janelia Fluor® 525 in as little at 90 minutes (15 minutes active time) with a high degree of labeling. There are many benefits of directly labeled proteins and antibodies such as:

  • Much easier multiplexing - no need to mix and match antibody species correctly
  • Avoid non-specific binding by secondary antibodies
  • Save time by shortening staining protocols.

Requirements

  • Not compatible with BSA containing antibodies or proteins as this will reduce the taget labeling. These should be removed before processing.
  • The protein to be labeled should be greater than 7kDa in size
  • The antibody concentration should be at least 1mg/ml, lower concentrations can be used however this will effect the degree of labeling.

Pack size guidance

Please note:

  • The 2x50µg packsize is sufficient to carry out 2 conjugation reactions on 50µg protein each.
  • The 3x100µg packsize is sufficent to carry out 3 conjugation reactions on 100µg protein each.
Description

Kit for conjugation of antibodies and other proteins to Janelia Fluor® 525

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Images

Figure 1. Janelia Fluor 525 conjugated anti-Myelin Basic Protein antibody staining in rat cerebellum

A mouse monoclonal anti-Myelin Basic Protein antibody (HB8014) was conjugated to Janelia Fluor 525 and used to stain rat cerebellum sections. Method: Rat brains were then dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in conjugated HB8014 (2µg/ml). DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as a z-stack in Lightning deconvolution mode using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope (40x objective, 405nm and 514nm lasers). The stack was subsequently flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 2. Janelia Fluor 525 conjugated anti-Myelin Basic Protein antibody staining in rat cerebellum

A mouse monoclonal anti-Myelin Basic Protein antibody (HB8014) was conjugated to Janelia Fluor 525 and used to stain rat cerebellum sections. Method: Rat brains were then dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in conjugated HB8014 (2µg/ml). DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as a z-stack in Lightning deconvolution mode using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope (20x objective, 405nm and 514nm lasers). The stack was subsequently flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 3. Janelia Fluor 525 conjugated anti-Myelin Basic Protein antibody staining in rat cerebellum

A mouse monoclonal anti-Myelin Basic Protein antibody (HB8014) was conjugated to Janelia Fluor 525 and used to stain rat cerebellum sections. Method: Rat brains were then dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in conjugated HB8014 (2µg/ml). DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as a tilescan and z-stack using a Leica DMI6000 inverted epifluorescence microscope (20x objective, DAPI and RHO filters). The tilescan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Optical Data

Fluorescence spectra Fluorescence Spectra
Emission color Yellow
Max excitation wavelength 525 nm
Max emission wavelength 549 nm
Closest laser lines 514nm
Spectrally similar dyes Alexa Fluor® 514, Alexa Fluor® 532, Atto 532, CF532, CF514
Quantum Yield (φ) 0.91
Extinction Coefficient (ε) 122,000 M-1cm-1

Biological Data

Application notes

Conjugation Protocol

Please follow this link to the conjugation protocol. Conjugation takes around 90 minutes with only 15 minutes of active time.

Solubility & Handling

Storage instructions

+4°C, except HB8455 at -20°C. Protect dye from moisture and light.

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use.

Calculators

Molarity

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More Info

Dilution

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More Info

Chemical Data

Kit contents
  • Lyophilised Janelia Fluor dye
  • DMSO
  • Lyophilised conjugation buffer
  • Lyophilised quenching buffer
  • Protein storage buffer
  • Microfuge desalting columns

FAQs

Can this conjugation kit be used with BSA containing antibodies?

This conjugation kit is not compatible with antibodies containing BSA in their storage buffer. This is because the concentration of BSA is much higher than the antibody meaning that using the standard protocol will result in an extremely low degree of labelling for the antibody. We recommend either purchasing BSA free antibodies or removing BSA from the buffer before conjugation.

How stable is the resulting fluorophore conjugated protein?

Because the conjugation reaction forms a covalent bond between the fluorophore and protein this results in a very stable conjugated protein. In our experience of conjugating antibodies, the fluorophore tagged antibodies are stable for at least a year at 4°C and much longer if stored correctly at either -20°C or -80°C.

References for Janelia Fluor® 525 conjugation kit

References are publications that support the biological activity of the product
  • A general method to fine-tune fluorophores for live-cell and in vivo imaging.

    Grimm JB et al (2017) Nature methods 14 : 987-994

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