Product overview
| Name | Streptavidin AF488 |
| Biological description | Streptavidin AF488 is a biotin binding protein conjugated with the fluorescent dye AF488 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 AF488 enabling immunofluorescent detection in IHC, ICC, flow cytometry and Western blot.
Key features: · Conjugated with AF488 (Ex: 498nm, Em: 525nm) · Supplied as a more stable lyophilate · Bright and photostable signal for repeated imaging · Suited for IHC(IF), ICC, Western blotting and Flow cytometry |
| Description | AF488 conjugated streptavidin for detection and signal amplification of biotin coupled proteins and antibodies. |
Images
Figure 1. Parvalbumin and Calretinin expressing interneurons 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 Parvalbumin HB6457 (1:1,000 dilution) and Calretinin HB6494 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotin antibody HB11345, Goat anti-rabbit Dylight 550, Invitrogen). Following three washes in PBST, sections were incubated with Streptavidin AF488 HB13531at 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 as a tilescan using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 524.211 ms, Y3: 524.211 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. Parvalbumin expressing interneurons and GFAP expressing astrocytes in the 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 Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotinylated antibody HB11345, Goat anti-chicken 647, Invitrogen). Following three washes in PBST, sections were incubated with XFD488 Streptavidin HB13531 at 1.0 µL/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: A4: 57.803 ms, GFP: 151.803 ms, Y5: 599.044 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 3. Concentration response of Streptavidin AF488 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 Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotin antibody HB11345). Following three washes in PBST, sections were incubated with Streptavidin AF488 (HB13531) at 1.0 µg/mL, 5.0 µg/mL and 10.0 gL/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our . 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: A4: 57.803 ms, GFP: 151.803 ms
5.0 µg/mL: A4: 71.225 ms, GFP: 381.278 ms
10.0 µg/mL: A4: 71.225 ms, GFP: 401.056 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: Parvalbumin expressing interneurons 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 Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:250 dilution of goat anti-mouse biotinylated antibody HB11345. Following three washes in PBST, sections were incubated with Streptavidin AF488 (HB13531) 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: A4: 50.000 ms, GFP: 104.469 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 and Calretinin expressing interneurons 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 Parvalbumin HB6457 (1:1,000 dilution) and Calretinin HB6494 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotin antibody HB11345, Goat anti-rabbit Dylight 550, Invitrogen). Following three washes in PBST, sections were incubated with Streptavidin AF488 HB13531at 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 as a tilescan using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 524.211 ms, Y3: 524.211 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. Parvalbumin expressing interneurons and GFAP expressing astrocytes in the 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 Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotinylated antibody HB11345, Goat anti-chicken 647, Invitrogen). Following three washes in PBST, sections were incubated with XFD488 Streptavidin HB13531 at 1.0 µL/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: A4: 57.803 ms, GFP: 151.803 ms, Y5: 599.044 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 3. Concentration response of Streptavidin AF488 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 Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:300 dilution (goat anti-mouse biotin antibody HB11345). Following three washes in PBST, sections were incubated with Streptavidin AF488 (HB13531) at 1.0 µg/mL, 5.0 µg/mL and 10.0 gL/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our . 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: A4: 57.803 ms, GFP: 151.803 ms
5.0 µg/mL: A4: 71.225 ms, GFP: 381.278 ms
10.0 µg/mL: A4: 71.225 ms, GFP: 401.056 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: Parvalbumin expressing interneurons 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 Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with secondary antibody at a 1:250 dilution of goat anti-mouse biotinylated antibody HB11345. Following three washes in PBST, sections were incubated with Streptavidin AF488 (HB13531) 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: A4: 50.000 ms, GFP: 104.469 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 | Green |
| Max excitation wavelength | 490 |
| Max emission wavelength | 525 |
| Closest laser lines | 488 nm |
| Spectrally similar dyes | Alexa Fluor® 488, Atto 488, DyLight® 488, FITC |
| Quantum Yield (φ) | 0.92 |
| Extinction Coefficient (ε) | 73000 M-1cm-1 |
| Correction Factor 280 | 0.11 |
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 AF488 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 either a 488nm laser or GFP filter set to excite Streptavidin AF488. |
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 |
References for Streptavidin AF488
-
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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