Streptavidin Janelia Fluor® 646
Product overview
| Name | Streptavidin Janelia Fluor® 646 |
| Biological description | Streptavidin Janelia Fluor® 646 is a biotin binding protein conjugated with the fluorescent dye Janelia Fluor® 646 and can be used to detect biotin labelled molecules such as nucleic acids, antibodies, and other proteins. Biotinylated antibodies are bound with extremely high affinity by Streptavidin Janelia Fluor® 646 enabling immunofluorescent detection in IHC, ICC, flow cytometry and Western blot. Janelia Fluor® 646 and the other members of the Janelia Fluor® family are bright and highly photostable fluorophores particularly suited for super resolution imaging such as dSTORM and STED.
Key features: · Conjugated with Janelia Fluor® 646 (Ex: 652nm, Em: 675nm) · Supplied as a more stable lyophilate · Bright and photostable signal for repeated imaging · Suited for IHC(IF), ICC, Western blotting and Flow cytometry |
| Description | Janelia Fluor® 646 conjugated streptavidin for detection and signal amplification of biotin coupled proteins and antibodies. |
Images
Figure 1. Parvalbumin positive interneurons and GFAP positive astrocytes in the rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250, and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: 1.0 µg/mL: DAP: 57.803 ms, GFP: 3061.716 ms, Y5: 1165.153 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calretinin and Parvalbumin expression in rat hippocampus.
Method: Rat brains were dissected and fixed overnight in 4% PFA before incubation 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% donkey serum before incubation overnight in anti-calretinin HB6494 (1:4,000 dilution) and anti-parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036) at a dilution of 1:250, and goat anti-mouse Dylight™ 594 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured using a 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 580nm (3.63% power, Hyd: 68% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-Parvalbumin HB6457 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL, 5.0 µg/mL or 10 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.5 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
- 1.0 µg/mL: DAP: 57.803 ms, Y5: 1165.153 ms
- 5.0 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
- 10.0 µg/mL: DAP: 139.756 ms, Y5: 431.117 ms
Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Calretinin positive interneurons and astrocytes in the rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-calretinin HB6494 (1:1,000 dilution) and anti-GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036 at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 488nm (2.18% power, Hyd: 22.9% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-parvalbumin HB6494 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL or 5.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.5 µg/mL: DAP: 46.156 ms, Y5: 11222.92 ms
- 1.0 µg/mL: DAP: 46.156 ms, Y5: 6131.079 ms
- 5.0 µg/mL: DAP: .756 ms, Y5: 11222.92 ms
Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Parvalbumin positive interneurons and GFAP positive astrocytes in the rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250, and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: 1.0 µg/mL: DAP: 57.803 ms, GFP: 3061.716 ms, Y5: 1165.153 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Calretinin and Parvalbumin expression in rat hippocampus.
Method: Rat brains were dissected and fixed overnight in 4% PFA before incubation 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% donkey serum before incubation overnight in anti-calretinin HB6494 (1:4,000 dilution) and anti-parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036) at a dilution of 1:250, and goat anti-mouse Dylight™ 594 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured using a 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 580nm (3.63% power, Hyd: 68% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-Parvalbumin HB6457 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL, 5.0 µg/mL or 10 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.5 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
- 1.0 µg/mL: DAP: 57.803 ms, Y5: 1165.153 ms
- 5.0 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
- 10.0 µg/mL: DAP: 139.756 ms, Y5: 431.117 ms
Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Calretinin positive interneurons and astrocytes in the rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-calretinin HB6494 (1:1,000 dilution) and anti-GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036 at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 488nm (2.18% power, Hyd: 22.9% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Method: Rat brains were 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% donkey serum before incubation overnight in anti-parvalbumin HB6494 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL or 5.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.5 µg/mL: DAP: 46.156 ms, Y5: 11222.92 ms
- 1.0 µg/mL: DAP: 46.156 ms, Y5: 6131.079 ms
- 5.0 µg/mL: DAP: .756 ms, Y5: 11222.92 ms
Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Optical Data
| Fluorescence spectra |  |
| Emission color | Red |
| Max excitation wavelength | 646 nm |
| Max emission wavelength | 664 nm |
| Closest laser lines | 640nm |
| Spectrally similar dyes | Alexa Fluor® 647, Atto 647, DyLight® 650, Cyanine 5 |
| Quantum Yield (φ) | 0.54 |
| Extinction Coefficient (ε) | 152000 M-1cm-1 |
| Correction Factor 280 | 0.19 |
Biological Data
| Application notes | #Protocol 1: Detecting biotin-labelled antibodies in IHC 1. Incubate free floating rat brain sections (40µm) in sodium borohydride (NaBH4) for 15 minutes followed by 2 hours in blocking buffer (0.05M glycine, 2% BSA and 3% donkey serum). 2. Incubate sections with primary antibody in blocking buffer at 4°C overnight, as in our IHC protocol. 3. Wash sections three times in PBST for 5 minutes each. 4. Incubate sections with 2 µg/mL goat anti-mouse biotin antibody HB11345 or goat anti-rabbit antibody HB11036 diluted in blocking buffer for 2 hours at RT. 5. Wash sections three times in PBST for 5 minutes each. 6. Incubate sections with 1 µg/mL Streptavidin Janelia Fluor® 646 in blocking buffer for 2 hours. 7. Wash sections three time in PBST for 5 minutes each. 8. Incubate sections with 10 µg/mL DAPI for 10 minutes. 9. Wash sections in dH2O, mount on glass slides with mounting media and cover with coverslip. 10. Image the sections on a microscope using a 640nm laser or Cy5 filter set to excite Streptavidin Janelia Fluor® 646. |
Solubility & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either 1ml of:
|
| Storage buffer | When reconstituted contains PBS with 0.05% sodium azide and 1% recombinant BSA. |
| 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
| Molecular Weight | 52 |
| Formulation | Lyophilised. When reconstituted contains 0.05% sodium azide and 1% recombinant albumin |
| Licensing details | Sold under license from the Howard Hughes Medical Institute, Janelia Research Campus |
References for Streptavidin Janelia Fluor® 646
-
Recent advances in the engineering and application of streptavidin-like molecules.
Le Q et al (2019) Applied microbiology and biotechnology 103 : 7355-7365 -
Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.
Dundas CM et al (2013) Applied microbiology and biotechnology 97 : 9343-53 -
Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.
Wilchek M et al (2006) Immunology letters 103 : 27-32
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