Alexa Fluor® 647 anti-Bcl-2 Antibody

Product Details

Verified Reactivity

Human

Antibody Type

Monoclonal

Host Species

Mouse

Immunogen

Synthetic peptide of amino acids 41-54 of human bcl-2 oncoprotein

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 Alexa Fluor® 647 under optimal conditions.

Concentration

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

ICFC - Quality tested
SB - Reported in the literature, not verified in house

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 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.

* 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.

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Excitation Laser

Red Laser (633 nm)

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. Pezzella F, et al. 1993. New Eng. J. Med. 329:690. (IHC)
2. Pezzella F, et al. 1990. Am. J. Pathol. 137:225. (IHC)
3. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
4. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Cabello CM, et al. 2009. Free Radic Biol Med. 220:46. PubMed
2. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
3. Lin J, Sorger M 2015. Sci Rep. 6: 8390. PubMed
4. Herrera FG, et al. 2019. Int J Radiat Oncol Biol Phys. 103:320. PubMed
5. Cai J, et al. 2021. eLife. 10:00. PubMed

RRID

AB_2563279 (BioLegend Cat. No. 658705)
AB_2563279 (BioLegend Cat. No. 658706)

Antigen Details

Structure

Two isoforms, 239 or 205 amino acids in length, with a predicted molecular weight of 26 kD or 22 kD and four ‘BH' domains.

Distribution

Outer mitochondrial membrane, nuclear membrane, endoplasmic reticulum membrane.

Function

Regulates apoptosis through controlling mitochondrial fusion and fission.

Interaction

BAX, BAD, BAK, Bcl-X(L),EI24,APAF1, BBC3, BCL2L1, BNIPL, MRPL41, TP53BP2, FKBP8 BAG1, RAF1, EGLN3, G0S2, and BOP.

Cell Type

B cells

Biology Area

Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Cell Cycle/DNA Replication, Chromatin Remodeling/Epigenetics, Immunology, Mitochondrial Function, Neuroscience, Signal Transduction, Transcription Factors, Ubiquitin/Protein Degradation

Antigen References

1. Lin P, et al. 2013. Curr. Hematol. Malig. Rep. 8:243.
2. Tomita N. 2011. J. Clin. Exp. Hematop. 51:7.
3. Kelly PN, et al. 2011. Cell Death Differ. 18:1414.
4. Willimott S, et al. 2010. Biochem. Soc. Trans. 38:1571.
5. Rolland SG, et al. 2010. Curr. Opin. Cell Biol. 22:852.

Gene ID

596 View all products for this Gene ID

UniProt

View information about Bcl-2 on UniProt.org

Documentation

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

Reviews

Related Protocols

• Surface and Intracellular Cytokine Staining for Flow Cytometry - Video
• Intracellular 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