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:
Requirements
Pack size guidance Please note:
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| 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).
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 |  |
| 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. |
Chemical Data
| Kit contents |
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Technical guides
Janelia Fluor Conjugation Kit ProtocolReferences for Janelia Fluor® 525 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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