Biolegend, 658706, Alexa Fluor® 647 anti-Bcl-2 Antibody, 100tests

Bcl-2 is a conserved anti-apoptotic protein that plays important roles in normal immunity. It forms homodimers or heterodimers with other Bcl-2 family members. This protein regulates apoptosis through controlling mitochondrial fusion and fission. Phosphorylation of Bcl-2 has been shown to enhance activity to allow response to extracellular growth-factor-mediated signals. Bcl-2 is also regulated by IRES, miR-15a, miR-16-1 and RNA-BP nucleolin. Chromosome 18q21.3 translocation and overexpression of Bcl-2 is frequently observed in follicular lymphomas and some diffuse large B-cell lymphomas.
100tests
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.View full statement regarding label licenses
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): Pezzella F, et al. 1993. New Eng. J. Med. 329:690. (IHC) Pezzella F, et al. 1990. Am. J. Pathol. 137:225. (IHC) 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: Cabello CM, et al. 2009. Free Radic Biol Med. 220:46. PubMed Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed Lin J, Sorger M 2015. Sci Rep. 6: 8390. PubMed Herrera FG, et al. 2019. Int J Radiat Oncol Biol Phys. 103:320. PubMed Cai J, et al. 2021. eLife. 10:00. PubMed
RRID: AB_2563279 (BioLegend Cat. No. 658705) AB_2563280 (BioLegend Cat. No. 658706)
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
UniProt: View information about Bcl-2 on UniProt.org
Clone: 100
Regulatory Status: RUO
Other Names: B-Cell CLL/Lymphoma 2 (Bcl-2), Protein Phosphatase 1, Regulatory Subunit 50
Isotype: Mouse IgG1
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.