Alexa Fluor® 647 anti-mouse Ly-6G Antibody

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

Ly-6G transfected EL-4J cell line.

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

FC - Quality tested
IHC-F - Verified
SB - Community verified

Recommended Usage

Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤ 0.25 µg per 10⁶ cells in 100 µl volume. 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 633nm / 635nm.

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

While 1A8 recognizes only Ly-6G, clone RB6-8C5 recognizes both Ly-6G and Ly-6C. Clone RB6-8C5 binds with high affinity to mouse Ly-6G molecules and to a lower extent to Ly-6C¹⁵. Clone RB6-8C5 impairs the binding of anti-mouse Ly-6G clone 1A8¹⁵. However, clone RB6-8C5 is able to stain in the presence of anti-mouse Ly-6C clone HK1.4¹⁶.

Additional reported applications (for the relevant formats) include: immunohistochemistry⁹ of frozen sections¹⁰ and paraffin-embedded sections¹¹, depletion^{4, 12-14}, and spatial biology (IBEX)^20,21. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for in vivo studies or highly sensitive assays (Cat. No. 127632, 127649, 127650, 127661 and 127662).

Additional Product Notes

This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) 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)

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13. Jaeger BN, et al. 2012. J. Exp. Med. 209:565. (Deplete)
14. Wozniak KL, et al. 2012. BMC Immunol. 13:65 (FC, Deplete)
15. Ribechini E, et al. 2009. Eur. J. Immunol. 39:3538.
16. Ng LG, et al. 2011. J Invest. Dermatol. 131:2058. PubMed
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20. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
21. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

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RRID

AB_1134159 (BioLegend Cat. No. 127609)
AB_1134159 (BioLegend Cat. No. 127610)

Antigen Details

Structure

A 21-35 kD GPI-anchorded membrane protein

Distribution

Expressed on the majority of myeloid cells in bone marrow and peripheral granulocytes. The monoclonal antibody RB6-8C5 recognizes both Ly-6G and Ly-6C.

Cell Type

Granulocytes, Macrophages, Monocytes

Biology Area

Immunology, Innate Immunity

Antigen References

Fleming TJ, et al. 1993. J. Immunol. 151:2399.

Gene ID

546644 View all products for this Gene ID

UniProt

View information about Ly-6G on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Immunohistochemistry Protocol for Frozen Sections
• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

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