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.
Figure 1. Parvalbumin and Calretinin expressing interneurons in the rat hippocampus.
Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6494 to detect Calretinin. Parvalbumin was detected using anti-mouse biotin antibody (HB11345 followed by incubation with Streptavidin AF488 (HB13531).
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.
Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6406 to detect GFAP. Parvalbumin was detected using anti-mouse biotin antibody (HB11345 followed by incubation with Streptavidin AF488 (HB13531).
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.
Streptavidin AF488 was used to detect Parvalbumin expressing interneurons in the rat hippocampus. Streptavidin AF488 (HB13531) produces strong staining down to concentrations as low as 1 µg/ml.
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:
Figure 4: Parvalbumin expressing interneurons in the rat hippocampus
Rat hippocampus stained with HB6457 to detect Parvalbumin. Parvalbumin was detected using an anti-mouse biotin antibody (HB11345) followed by incubation with Streptavidin AF488 (HB13531).
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.
Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6494 to detect Calretinin. Parvalbumin was detected using anti-mouse biotin antibody (HB11345 followed by incubation with Streptavidin AF488 (HB13531).
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.
Rat hippocampus stained with HB6457 to detect Parvalbumin and HB6406 to detect GFAP. Parvalbumin was detected using anti-mouse biotin antibody (HB11345 followed by incubation with Streptavidin AF488 (HB13531).
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.
Streptavidin AF488 was used to detect Parvalbumin expressing interneurons in the rat hippocampus. Streptavidin AF488 (HB13531) produces strong staining down to concentrations as low as 1 µg/ml.
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:
Figure 4: Parvalbumin expressing interneurons in the rat hippocampus
Rat hippocampus stained with HB6457 to detect Parvalbumin. Parvalbumin was detected using an anti-mouse biotin antibody (HB11345) followed by incubation with Streptavidin AF488 (HB13531).
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:
dH2O and storing at 4°C
50:50 ratio of dH2O to glycerol and storing at -20°C
dH2O then aliquot and store at -80°C
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the streptavidin is thoroughly dissolved by pipetting up and down before giving the streptavidin a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube.
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