Product overview
| Name | Janelia Fluor® 549 conjugation kit |
| Biological description | Overview The Hello Bio Janelia Fluor® 549 conjugation kit allows the conjugation of antibodies and proteins to Janelia Fluor® 549 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:
Requirements
Pack size guidance Please note:
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| Description | Kit for conjugation of antibodies and other proteins to Janelia Fluor® 549 |
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Images
Figure 1. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat hippocampus
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat hippocampal 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 HB6498 (4µg/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 using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI / TX2 filters. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat cortex.
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat cortical 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 HB6498 (4µg/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 using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI / TX2 filters. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat brain
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat brain 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 HB6498 (4µg/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 using a Leica SPE confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Janelia Fluor® 549 conjugated secondary antibodies show superior antifade performance compared to those conjugated with DyLight 550
Janelia Fluor® 549 conjugated secondary antibodies show greater resistance to photobleaching than DyLight 550 conjugated secondaries in a HEK293T based cell assay. Method: HEK293T cells were cultured on 10mm coverslips and stained with HB6491 (1:8,000 dilution, 125ng/ml) and either a goat anti-mouse DyLight 550 or goat anti-mouse Janelia Fluor® 549 conjugated secondary antibody. For more information please see our ICC protocol. Imaging was conducted with a Leica SPE confocal laser scanning microscope where cells were repeatedly imaged with the 488nm laser at 100% power over 30 seconds of continuous exposure to induce photobleaching. Average intensity values were calculated from images then data was normalised to the first exposure frame to generate normalised intensity. Statistics: decay curve: F-test, F5,350=1685, p<0.0001, final intensity: two tailed t-test, t16=19.15, p<0.0001.
Figure 1. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat hippocampus
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat hippocampal 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 HB6498 (4µg/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 using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI / TX2 filters. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat cortex.
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat cortical 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 HB6498 (4µg/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 using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI / TX2 filters. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Janelia Fluor 549 conjugated anti-NeuN antibody staining in rat brain
A rabbit polyclonal anti-NeuN antibody (HB6498) was conjugated to Janelia Fluor 549 and used to stain rat brain 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 HB6498 (4µg/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 using a Leica SPE confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Janelia Fluor® 549 conjugated secondary antibodies show superior antifade performance compared to those conjugated with DyLight 550
Janelia Fluor® 549 conjugated secondary antibodies show greater resistance to photobleaching than DyLight 550 conjugated secondaries in a HEK293T based cell assay. Method: HEK293T cells were cultured on 10mm coverslips and stained with HB6491 (1:8,000 dilution, 125ng/ml) and either a goat anti-mouse DyLight 550 or goat anti-mouse Janelia Fluor® 549 conjugated secondary antibody. For more information please see our ICC protocol. Imaging was conducted with a Leica SPE confocal laser scanning microscope where cells were repeatedly imaged with the 488nm laser at 100% power over 30 seconds of continuous exposure to induce photobleaching. Average intensity values were calculated from images then data was normalised to the first exposure frame to generate normalised intensity. Statistics: decay curve: F-test, F5,350=1685, p<0.0001, final intensity: two tailed t-test, t16=19.15, p<0.0001.
Optical Data
| Fluorescence spectra |  |
| Emission color | Yellow |
| Max excitation wavelength | 549nm |
| Max emission wavelength | 571nm |
| Closest laser lines | 561nm |
| Spectrally similar dyes | DyLight 550, Alexa Fluor® 546, Alexa Fluor® 555, BDY TMR-X, Atto 550, Cyanine 3, CF 555, TAMRA |
| Quantum Yield (φ) | 0.88 |
| Extinction Coefficient (ε) | 101,000 M-1cm-1 |
| Correction Factor 280 | 0.169 |
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 HB7336 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. |
Chemical Data
| Kit contents |
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Technical guides
Janelia Fluor Conjugation Kit ProtocolReferences for Janelia Fluor® 549 conjugation kit
References are publications that support the biological activity of the product
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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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