Anti-MAP2 antibody ValidAbTM
(HB9587)
Product overview
| Name | Anti-MAP2 antibody ValidAbTM |
| Host | Mouse |
| Clonality | Monoclonal |
| Target | MAP2 |
| Description | Antibody to MAP2 - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the cell body, dendrites and axon of a cultured neurone. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective (4.35x zoom), 405nm (45.6% power) and 488nm (31.2% power) laser lines in a z-stack (0.335 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the a dense network of culture neurones. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 40x objective (1x zoom), 405nm (45.6% power) and 488nm (31.2% power) laser lines in a z-stack (0.43 µm spacing). Deconvolution was carried out using Huygens Essential version (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the a dense network of culture neurones. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 36.2ms exposure) and I3 (3.4x gain, 150ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 4. MAP2 expression in rat CA1 pyramidal neurones visualised using HB9587.
HB9587 visualised the dense network of pyramidal neurone dendrites alongside interneurons of the stratum radiatum of CA1. Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Section were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in HB9587 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). 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 (20x objective) coupled to a Photometric-Prime95B monochrome digital camera with DAPI (10.2ms exposure) and L5 (112.7ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 5. The effect of varying HB9587 concentration upon staining in cultured rat neurones.
HB9587 produced a strong signal to noise ratio at dilutions as low as 1in2000 (500 ng/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at dilutions ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml) with a no primary step omitting the addition of HB9587 to the incubation buffer. This was followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and G/R filters. Exposure times were as follows:
- 1:500 – DAPI 56.6ms exposure, G/R 71.2ms exposure (captured on a different microscope – Leica DMI6000B, 40x objective, Photometric-Prime95B monochrome camera).
- 1:1000 – DAPI 1x gain, 43.7ms exposure, G/R 4.4x gain, 177.4ms exposure
- 1:2000 – DAPI 1x gain, 46.5ms exposure, G/R 4.5x gain, 210.3ms exposure
- 1:4000 – DAPI 1x gain, 40.0ms exposure, G/R 5.4x gain, 471.4ms exposure
- No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera).
Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool followed by stacking and montage creation.
Figure 6. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualising a cultured rat neurone. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (5.9x gain, 40.0ms exposure) and I3 (5.9x gain, 146.0ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 7. MAP2 expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions. 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 4-20% acrylamide gel alongside a protein ladder (NEB Prestained) before being run at 150V for 50 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 HB9587 at a 1:1000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 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 visualized again using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure).
Figure 8. MAP2 expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions. 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 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 41 minutes followed by 120V for 85 minutes. Wet transfer to a PVDF membrane was completed in 93 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9587 at a 1:1000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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).
Figure 9. Concentration response of HB9587 staining in a rat brain cytosol preparation.
HB9587 shows consistent results with low background at dilutions as low as 1:4,000 (250 ng/ml). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 30 minutes followed by 120V 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 HB9587. Each strip was incubated separately with a separate HB9587 concentration with this ranging from 2µg/ml (1:500 dilution) to 31.3ng/ml (1:32,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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. MAP2 and EGFR expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions whereas ab52894 (Abcam) revealed bands in P2 and liver 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 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 41 minutes followed by 120V for 85 minutes. Wet transfer to a PVDF membrane was completed in 93 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9587 at a 1:1000 dilution (1µg/ml) and ab52894 (Abcam, rabbit monoclonal) at a 1:2,000 dilution (concentration unavailable). Following washing the membrane was incubated in secondary antibodies (1:10,000 dilution of both polyclonal goat anti-mouse HRP conjugated (Sigma Aldrich A3682) and polyclonal goat anti-rabbit HRP conjugated (Sigma Aldrich 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).
Figure 1. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the cell body, dendrites and axon of a cultured neurone. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective (4.35x zoom), 405nm (45.6% power) and 488nm (31.2% power) laser lines in a z-stack (0.335 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the a dense network of culture neurones. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 40x objective (1x zoom), 405nm (45.6% power) and 488nm (31.2% power) laser lines in a z-stack (0.43 µm spacing). Deconvolution was carried out using Huygens Essential version (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualised the a dense network of culture neurones. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 36.2ms exposure) and I3 (3.4x gain, 150ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 4. MAP2 expression in rat CA1 pyramidal neurones visualised using HB9587.
HB9587 visualised the dense network of pyramidal neurone dendrites alongside interneurons of the stratum radiatum of CA1. Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Section were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in HB9587 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). 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 (20x objective) coupled to a Photometric-Prime95B monochrome digital camera with DAPI (10.2ms exposure) and L5 (112.7ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 5. The effect of varying HB9587 concentration upon staining in cultured rat neurones.
HB9587 produced a strong signal to noise ratio at dilutions as low as 1in2000 (500 ng/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at dilutions ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml) with a no primary step omitting the addition of HB9587 to the incubation buffer. This was followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and G/R filters. Exposure times were as follows:
- 1:500 – DAPI 56.6ms exposure, G/R 71.2ms exposure (captured on a different microscope – Leica DMI6000B, 40x objective, Photometric-Prime95B monochrome camera).
- 1:1000 – DAPI 1x gain, 43.7ms exposure, G/R 4.4x gain, 177.4ms exposure
- 1:2000 – DAPI 1x gain, 46.5ms exposure, G/R 4.5x gain, 210.3ms exposure
- 1:4000 – DAPI 1x gain, 40.0ms exposure, G/R 5.4x gain, 471.4ms exposure
- No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera).
Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool followed by stacking and montage creation.
Figure 6. MAP2 expression in cultured rat neurones visualised using HB9587.
HB9587 visualising a cultured rat neurone. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB9587 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (5.9x gain, 40.0ms exposure) and I3 (5.9x gain, 146.0ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 7. MAP2 expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions. 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 4-20% acrylamide gel alongside a protein ladder (NEB Prestained) before being run at 150V for 50 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 HB9587 at a 1:1000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 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 visualized again using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure).
Figure 8. MAP2 expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions. 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 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 41 minutes followed by 120V for 85 minutes. Wet transfer to a PVDF membrane was completed in 93 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9587 at a 1:1000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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).
Figure 9. Concentration response of HB9587 staining in a rat brain cytosol preparation.
HB9587 shows consistent results with low background at dilutions as low as 1:4,000 (250 ng/ml). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 30 minutes followed by 120V 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 HB9587. Each strip was incubated separately with a separate HB9587 concentration with this ranging from 2µg/ml (1:500 dilution) to 31.3ng/ml (1:32,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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. MAP2 and EGFR expression in various tissue lysates and preparations.
HB9587 revealed a band of size greater than 250kDa only present in brain cytosol fractions whereas ab52894 (Abcam) revealed bands in P2 and liver 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 5% acrylamide gel alongside a protein ladder (BioRad Precision Plus dual colour, 1610374) before being run at 60V for 41 minutes followed by 120V for 85 minutes. Wet transfer to a PVDF membrane was completed in 93 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9587 at a 1:1000 dilution (1µg/ml) and ab52894 (Abcam, rabbit monoclonal) at a 1:2,000 dilution (concentration unavailable). Following washing the membrane was incubated in secondary antibodies (1:10,000 dilution of both polyclonal goat anti-mouse HRP conjugated (Sigma Aldrich A3682) and polyclonal goat anti-rabbit HRP conjugated (Sigma Aldrich 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).
Product information
| Immunogen | Microtubule preparation derived from bovine brain and enriched for kinesin |
| Epitope | Localised to within amino acids 1375 to 1395 (CPPAVSEADLATDERADVQME) |
| Clone number | MT-07 |
| Isotype | IgG1 |
| Purification | Protein A affinity chromatography |
| Concentration | 1mg/ml |
| Formulation | Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant BSA |
| Predicted species reactivity | Human, Mouse, Pig, Cow |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | ICC, WB, IHC(IF) |
| Western blot optimal concentration | 1µg/ml (1:1000) as measured in rat brain cytosol |
| IHC(IF) optimal concentration | 1µg/ml (1:1000) as measured in rat hippocampal sections |
| ICC optimal concentration | 1µg/ml (1:1000) as measured in cultured rat neurones |
| Positive control | MAP2 should be found in any neural tissue sample but is not widely expressed in cell lines. |
| Negative control | Non-neural tissues such as liver or muscle. Most common non-neural derived cell lines, such as HeLa and HEK293 are also MAP2 negative. |
| Open data link | Please follow this link to OSF |
Target information
| Other names | MAP-2, Microtubule-associated protein 2 |
| UniProt ID | P11137 |
| Structure image |  |
| Gene name | MAP2 |
| NCBI full gene name | microtubule associated protein 2 |
| Entrez gene ID |
| Amino acids | 1827 (199.5kDa) |
| Isoforms | MAP2 has 4 key isoforms: Isoform 1 (MAP2b), 1827aa, 199.5kDa; Isoform 2 (MAP2c), 471aa, 49.6kDa, missing aa152-1507 - juvenile isoform not expressed in adulthood; Isoform 3, 1823aa, 199.0kDa, missing aa152-155; Isoform 4, 559aa, 59.0kDa, multiple substitutions and missing aa230-1528. |
| Expression | Expressed highly within the brain (neuron specific) and to a lesser degree in the testes |
| Subcellular expression | Expressed as part of the cytoskeleton |
| Processing | None |
| Post translational modifications | MAP2 contains numerous phosphorylation sites however none occur with the epitope of HB9587 |
| Homology (compared to human) | Mouse and rat show 79.8% and 77.7% identity to human MAP2 respectively in a BLAST search. |
| Similar proteins | None |
| Epitope homology (between species) | Human, 100% identity Mouse, 76.2% identity Rat, 71.4% identity |
| Epitope homology (other proteins) | No significant homology with other proteins |
Storage & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either:
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube. For more information please see our detailed guide on storing and using your antibody |
| 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) References for Anti-MAP2 antibody ValidAbTM
References are publications that support the biological activity of the product
-
The MAP2/Tau family of microtubule-associated proteins
Dehmelt L and Halpain S (2005) Genome Biol 6(1) : 204 -
Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons
Chen J et al (1992) Nature 360(6405) : 674-677 -
Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein
Lewis SA, Wang DH and Cowan NJ (1988) Science 242(4880) : 936-939 -
Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain
Huber G, Matus A (1984) Journal of Neuroscience 4(1) : 151-160
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