Biolegend, 657412, Brilliant Violet 421™ anti-Tubulin Beta 3 (TUBB3) Antibody, 100μg

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.
100μg
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 and BSA (origin USA).
Preparation: The antibody was purified by affinity chromatography and conjugated with Brilliant Violet 421™ under optimal conditions.
Concentration: 0.2 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: IHC-F - Quality tested ICC - Verified SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunohistochemical staining on frozen tissue sections. For immunohistochemical staining on frozen tissue sections, the suggested use of this reagent is 1.25 - 5.0 µg/ml. For immunocytochemistry, a concentration range of 1.25 - 5.0 μg/ml is recommended. It is recommended that the reagent be titrated for optimal performance for each application. Brilliant Violet 421™ excites at 405 nm and emits at 421 nm. The standard bandpass filter 450/50 nm is recommended for detection. Brilliant Violet 421™ is a trademark of Sirigen Group Ltd. Learn more about Brilliant Violet™. This product is subject to proprietary rights of Sirigen Inc. and is made and sold under license from Sirigen Inc. The purchase of this product conveys to the buyer a non-transferable right to use the purchased product for research purposes only. This product may not be resold or incorporated in any manner into another product for resale. Any use for therapeutics or diagnostics is strictly prohibited. This product is covered by U.S. Patent(s), pending patent applications and foreign equivalents.
Excitation Laser: Violet Laser (405 nm)
Application Notes: Additional reported application (for relevant formats) include: spatial biology (IBEX)1,2.
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
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
RRID: AB_2632699 (BioLegend Cat. No. 657412)
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: What is the F/P ratio range of our BV421™ format antibody reagents?
A: It is lot-specific. On average it ranges between 2-4.
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.