Biolegend, 657405, Alexa Fluor® 647 anti-Tubulin Beta 3 (TUBB3) Antibody, 25μg

Tubulin is the main component of microtubules. In adults, tubulin beta 3 (TUBB3) is primarily expressed in neurons and is commonly used as a neuronal marker. It plays an important role in neuronal cell proliferation and differentiation. Mutations in this gene cause congenital fibrosis of the type 3 extraocular muscles. Tubulin beta 3 (TUBB3) is also found in a wide range of tumors. Studies indicate that it is a predictive and prognostic marker in various tumors.
25μg
Verified Reactivity: Mouse, Rat, Human
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: Fusion protein
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: 0.5 mg/mL
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: ICFC - Quality tested IHC-F, 3D IHC - Verified SB - Community verified
Recommended Usage: Each lot of this antibody is quality control tested by intracellular immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤ 0.06 µg per million cells in 100 µL volume. For immunohistochemical staining on frozen tissue sections, the suggested use is 1.25-5 µg/mL. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Red Laser (633 nm)
Application Notes: Additional reported application (for relevant formats) include: spatial biology (IBEX)1,2.
Additional Product Notes: This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) and IHC-P (Immunohistochemistry - formalin-fixed paraffin-embedded tissues) on the NanoString GeoMx® Digital Spatial Profiler. The GeoMx® enables researchers to perform spatial analysis of protein and RNA targets in FFPE and fresh frozen human and mouse samples. For more information about our spatial biology products and the GeoMx® platform, please visit our spatial biology page.
Application References(PubMed link indicates BioLegend citation): Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Khan NZ, et al. 2021. Brain Behav Immun. 92:165. PubMed Lin YC, et al. 2021. Nat Neurosci. 24:1077. PubMed Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed Buljan M, et al. 2020. Molecular Cell. 79(3):504-520. PubMed
RRID: AB_2563609 (BioLegend Cat. No. 657405) AB_2563610 (BioLegend Cat. No. 657406)
Structure: 450 amino acids with predicted molecular weight of 50 kD
Distribution: Cytosol
Function: Plays important roles in neuronal cell proliferation and differentiation
Interaction: Alpha tubulin, kinesin and dynein
Cell Type: Mature Neurons
Biology Area: Cell Biology, Cell Cycle/DNA Replication, Cell Motility/Cytoskeleton/Structure, Immunology, Neuroscience, Neuroscience Cell Markers
Molecular Family: Microtubules
Antigen References: 1. Katsetos CD, et al. 2003. J. Child Neurol. 18:851. 2. Mobarakeh ZT, et al. 2012. Cell Biol. Int. Rep. (2010) 19:e00015. 3. Locher H, et al. 2013. Differentiation. 85:173. 4. Karki R, et al. 2013. Expert Opin. Ther. Targets. 17:461. 5. Mariani M, et al. 2011. Curr. Mol. Med. 11:726. 6. Koh Y, et al. 2009. Ann. Oncol. 20:1414.
Gene ID: 10381
UniProt: View information about Tubulin beta-3 on UniProt.org
Clone: AA10
Regulatory Status: RUO
Other Names: β-3 Tubulin, Neuronal class III beta-tubulin, β3-tub
Isotype: Mouse IgG2a, κ
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.