Anti-Vesicular GABA transporter (VGAT) antibody ValidAb™
(HB6714)
Product overview
| Name | Anti-Vesicular GABA transporter (VGAT) antibody ValidAb™ |
| Host | Rabbit |
| Clonality | Polyclonal |
| Target | VGAT |
| Description | Antibody to VGAT - GABA transporter and GABAergic neuron marker. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Concentration response of HB6714 staining in rat cerebellum.
HB617 stains VGAT positive terminals at dilutions down to 1:4,000 (62.5ng/ml) – please note the concentration of HB6457 (mouse monoclonal anti-parvalbumin) is fixed at 0.5µg/ml (1:2,000 dilution). 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 HB6457 (1:2000 dilution, 0. 5µg/ml) and dilutions of HB6714 ranging from 1:500 to 1:4,000. This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The images were captured using a 20x objective in a z-stack and were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
- 1:500, DAPI: 20ms, Y5: 250ms,
- 1:1,000, DAPI: 31.6ms, Y5: 372.1ms
- 1:2,000, DAPI: 25ms, Y5: 609.4ms
- 1:4,000, DAPI: 25ms, Y5: 940.3ms
Figure 3. VGAT, Parvalbumin and GFAP expression in rat hippocampal CA1.
Rat CA1 stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:2000 dilution, 0. 5µg/ml) and HB6714 (1:500 dilution, 0. 5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 63.9ms, GFP: 73.9ms, Y3: 133.6ms, Y5: 372.1ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Whole cerebellum section stained for VGAT, Parvalbumin and GFAP.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:2000 dilution, 0. 5µg/ml) and HB6714 (1:500 dilution, 0. 5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 20.0ms, GFP: 60.0ms, Y3: 110.0ms, Y5: 250.0ms exposures) in a z-stack (4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 100x objective (DAPI: 7.5ms, GFP: 63.9ms, Y3: 43.1ms, Y5: 185.9s exposures) in a z-stack (0.2µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. VGAT expression in various tissue lysates and preparations.
HB6714 revealed the ≈56kDa band associated with VGAT only in neural tissue samples. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 30 minutes followed by 180V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714 at a 1:1,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure).
Figure 8. VGAT expression in various tissue lysates and preparations.
HB6714 revealed the ≈59kDa band associated with VGAT only in neural tissue samples. An additional band at ≈45 kDa was visualised which was tentatively identified as a possible cross-reactivity with the transcription factor Pou4f1 (43.3kDa). However, this cross-reactivity was not observed in other experiments. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714 at a 1:1,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using ChemiHRP ECL substrate (MossBio) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 9. Concentration response of HB6714 staining in a rat brain P2 membrane preparation.
HB6714 produces reliable results down to a 125ng/ml concentration (1:2,000 dilution). Method: P2 membrane fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 35 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714. Each strip was incubated separately with a separate HB6714 concentration with this ranging from 1:250 to 1:8,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 10. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Concentration response of HB6714 staining in rat cerebellum.
HB617 stains VGAT positive terminals at dilutions down to 1:4,000 (62.5ng/ml) – please note the concentration of HB6457 (mouse monoclonal anti-parvalbumin) is fixed at 0.5µg/ml (1:2,000 dilution). 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 HB6457 (1:2000 dilution, 0. 5µg/ml) and dilutions of HB6714 ranging from 1:500 to 1:4,000. This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The images were captured using a 20x objective in a z-stack and were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
- 1:500, DAPI: 20ms, Y5: 250ms,
- 1:1,000, DAPI: 31.6ms, Y5: 372.1ms
- 1:2,000, DAPI: 25ms, Y5: 609.4ms
- 1:4,000, DAPI: 25ms, Y5: 940.3ms
Figure 3. VGAT, Parvalbumin and GFAP expression in rat hippocampal CA1.
Rat CA1 stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:2000 dilution, 0. 5µg/ml) and HB6714 (1:500 dilution, 0. 5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 63.9ms, GFP: 73.9ms, Y3: 133.6ms, Y5: 372.1ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Whole cerebellum section stained for VGAT, Parvalbumin and GFAP.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:2000 dilution, 0. 5µg/ml) and HB6714 (1:500 dilution, 0. 5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 20.0ms, GFP: 60.0ms, Y3: 110.0ms, Y5: 250.0ms exposures) in a z-stack (4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 100x objective (DAPI: 7.5ms, GFP: 63.9ms, Y3: 43.1ms, Y5: 185.9s exposures) in a z-stack (0.2µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. VGAT expression in various tissue lysates and preparations.
HB6714 revealed the ≈56kDa band associated with VGAT only in neural tissue samples. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 30 minutes followed by 180V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714 at a 1:1,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure).
Figure 8. VGAT expression in various tissue lysates and preparations.
HB6714 revealed the ≈59kDa band associated with VGAT only in neural tissue samples. An additional band at ≈45 kDa was visualised which was tentatively identified as a possible cross-reactivity with the transcription factor Pou4f1 (43.3kDa). However, this cross-reactivity was not observed in other experiments. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714 at a 1:1,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using ChemiHRP ECL substrate (MossBio) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 9. Concentration response of HB6714 staining in a rat brain P2 membrane preparation.
HB6714 produces reliable results down to a 125ng/ml concentration (1:2,000 dilution). Method: P2 membrane fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 35 minutes followed by 130V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6714. Each strip was incubated separately with a separate HB6714 concentration with this ranging from 1:250 to 1:8,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma, A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 10. VGAT, Calbindin and GFAP expression in rat cerebellum.
Rat cerebellum stained by HB6406 for GFAP (chicken polyclonal), HB6396 for Calbindin (mouse monoclonal) and HB6714 for VGAT (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6396 (1:2000 dilution) and HB6714 (1:1000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 25ms, GFP: 163.7ms, Y3: 373.1ms, Y5: 1.0s exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | Synthetic peptide of N-terminal rat VGAT residues conjugated to keyhole limpet hemocyanin (KLH). |
| Isotype | IgG |
| Purification | Immunogen affinity chromatography |
| Concentration | 0.25 mg/ml |
| Formulation | 10 mM HEPES (pH 7.5), 150 mM NaCl, 100µg/ml BSA, and 50% glycerol. |
| Predicted species reactivity | Mouse, Rat, Dog, Chicken, Cow, Monkey |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | WB, IHC(IF) |
| Western blot optimal concentration | 0.25µg/ml (1:1,000) as tested in a rat brain P2 membrane fraction preparation. |
| IHC(IF) optimal concentration | 0.25µg/ml (1:1,000) as tested in paraformaldehyde fixed free-floating rat brain sections. |
| Positive control | VGAT is expressed widely across all brain regions in GABAergic neurones. |
| Negative control | VGAT expression is absent from most non-neural tissues (including the liver and muscle) and the vast majority of human cell lines (e.g. HEK293T and HeLa) |
| Open data link | Please follow this link to OSF. |
Target information
| Other names | SLC32A1, VIAAT, Vesicular inhibitory amino acid transporter |
| UniProt ID | Q9H598 |
| Structure image |  |
| Gene name | SLC32A1 |
| NCBI full gene name | solute carrier family 32 member 1 |
| Entrez gene ID |
| Amino acids | 525 (57.4kDa) |
| Isoforms | VGAT has only one described isoform |
| Expression | VGAT is expressed in GABAergic interneurons and glycinergic neurons in various regions of the central nervous system (CNS), including the cortex, hippocampus, cerebellum, and spinal cord. Additionally, VGAT is also expressed in some non-neuronal cells, such as pancreatic beta cells, where it plays a role in the release of GABA as a neurotransmitter or a paracrine signaling molecule. |
| Subcellular expression | VGAT expression is localised to synapses and is not expressed in the cell bodies, axons or dendrites of neurones. |
| Processing | VGAT is not subject to any processing in order to form an active conformation |
| Post translational modifications | VGAT is subject to phosphorylation on S98 and nitration on Y186. |
| Homology (compared to human) | Mouse and rat VGAT show 98.5% identity to human VGAT. Mouse and rat VGAT homologues show 99.6% identity (A77P and L384I) |
| Similar proteins | No similar proteins to VGAT were identified in a BLAST search. |
Storage & Handling
| Storage instructions | -20°C |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Anti-Vesicular GABA transporter (VGAT) antibody ValidAb™
References are publications that support the biological activity of the product
-
Vesicular GABA transporter (VGAT) transports β-alanine.
Juge N et al (2013) Journal of neurochemistry 127 : 482-6 -
The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice.
Saito K et al (2010) Molecular brain 3 : 40 -
Vesicular inhibitory amino acid transporter is a Cl-/gamma-aminobutyrate Co-transporter.
Juge N et al (2009) The Journal of biological chemistry 284 : 35073-8 -
Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system.
Bedet C et al (2000) Journal of neurochemistry 75 : 1654-63 -
The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons.
Chaudhry FA et al (1998) The Journal of neuroscience : the official journal of the Society for Neuroscience 18 : 9733-50
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