Product overview

Name Anti-MAP2 antibody ValidAbTM
Description

Antibody to MAP2 - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies.

Host Mouse
Clonality Monoclonal
Target MAP2
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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 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 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. 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 8. 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 9. 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. 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  Chemical Structure
Gene name MAP2
NCBI full gene name microtubule associated protein 2
Entrez gene ID

4133

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:


  • 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 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

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

FAQs

What other neuroscience markers are available?
Why wasn’t it possible to work out the molecular weight of MAP2 in a Western Blot?

As MAP2 was significantly heavier than the highest available protein marker then this means it isn’t possible to accurately calculate its molecular weight. For more information about calculating protein weights please see our guide on analysing Western blots.

What guarantee do you have that my MAP2 antibody will perform as expected?

We guarantee that your MAP2 antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

Will my MAP2 antibody work against species that have not been listed on the datasheet?

A species not being listed doesn’t mean that the MAP2 antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!

What protocols are available for use with this MAP2 antibody

We have made a comprehensive collection of protocols that we have used in our experiments to validate this MAP2 antibody.

What counterstains do you recommend for use in ICC and IHC with this MAP2 antibody?

We recommend using either DAPI or Hoechst 33342 to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies