Brilliant Violet 421™ anti-mouse CD45 Antibody

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

Mouse thymus or spleen

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

µg sizes: 0.2 mg/mL
µL sizes: lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

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

FC - Quality tested

SB - Reported in the literature, not verified in house

Recommended Usage

Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining using the µg size, the suggested use of this reagent is ≤0.25 µg per million cells in 100 µl volume. For immunofluorescent staining using the µl size, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. 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

Clone 30-F11 reacts with all isoforms and both CD45.1 and CD45.2 alloantigens of CD45.

Additional reported applications (for relevant formats) include: immunoprecipitation³, complement-dependent cytotoxicity^1,5, immunohistochemistry (acetone-fixed frozen sections, zinc-fixed paraffin-embedded sections and formalin-fixed paraffin-embedded sections)^4,6, Western blotting⁷, and spatial biology (IBEX)^10,11. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 103163 and 103164).

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)

1. Podd BS, et al. 2006. J. Immunol. 176:6532. (FC, CMCD) PubMed
2. Haynes NM, et al. 2007. J. Immunol. 179:5099. (FC)
3. Ledbetter JA, et al. 1979. Immunol. Rev. 47:63. (IP)
4. Simon DI, et al. 2000. J. Clin. Invest. 105:293. (IHC)
5. Seaman WE. 1983. J. Immunol. 130:1713. (CMCD)
6. Cornet A, et al. 2001. P. Natl. Acad. Sci. USA 98:13306. (IHC)
7. Tsuboi S and Fukuda M. 1998. J. Biol. Chem. 273:30680. (WB) PubMed
8. Liu F, et al. 2012. Blood. 119:3295. PubMed
9. Pelletier AN, et al. 2012. J. Immunol. 188:5561. PubMed
10. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
11. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

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RRID

AB_10899570 (BioLegend Cat. No. 103133)
AB_10899570 (BioLegend Cat. No. 103134)

Antigen Details

Structure

Protein tyrosine phosphatase (PTP) family, 180-240 kD

Distribution

All hematopoietic cells except mature erythrocytes and platelets

Function

Phosphatase, T and B cell activation

Ligand/Receptor

Galectin-1, CD2, CD3, CD4, TCR, CD22, Thy-1

Cell Type

B cells, Dendritic cells, Mesenchymal Stem Cells, Tregs

Biology Area

Cell Biology, Immunology, Inhibitory Molecules, Innate Immunity, Neuroscience, Neuroscience Cell Markers, Stem Cells

Molecular Family

CD Molecules

Antigen References

1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press.
2. Trowbridge IS, et al. 1993. Annu. Rev. Immunol. 12:85.
3. Kishihara K, et al. 1993. Cell 74:143.
4. Pulido R, et al. 1988. J. Immunol. 140:3851.

Gene ID

19264 View all products for this Gene ID

UniProt

View information about CD45 on UniProt.org

Documentation

• Technical data sheet
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

What is the F/P ratio range of our BV421™ format antibody reagents?

It is lot-specific. On average it ranges between 2-4.

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

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.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

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.

Are other fluorophores compatible with IBEX?

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.

The same antibody works in one tissue type but not another. What is happening?

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.

How can I be sure the staining I’m seeing in my tissue is real?

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.

Other Formats

Certificate of Analysis