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
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).