ABCG19 Antibody

What is ABCG2 and why is it significant in cancer and stem cell research?

ABCG2 (ATP-binding cassette gene 2), also known as Bcrp1 (Breast cancer resistance protein 1), is a membrane transporter molecule first identified in a breast cancer cell line and subsequently found to be expressed on stem cells . It belongs to a family of molecules that span the cell membrane six times and can exist as either homo or hetero dimers linked by a short intracellular flexible linker region. This transporter plays a crucial role in the efflux of a wide range of substrates .

Beyond its initially recognized role in drug resistance, ABCG2 has become valuable for characterizing primitive stem cells. The "side-population" of hematopoietic stem cells, characterized by their inability to retain high levels of intracellular staining dyes Hoechst 33342 and Rhodamine 123, expresses high levels of ABCG2. This function has been directly linked to the efflux of the Hoechst dye . Furthermore, ABCG2 serves as a cell surface marker for identifying hematopoietic stem cells within the bone marrow fraction of lineage negative cells.

What applications are typically used for ABCG2 antibodies in research?

ABCG2 antibodies are predominantly utilized in the following applications:

• Flow cytometry for detection of ABCG2 expression in various cell lines, including cancer cells like MCF-7 human breast cancer cells

• Identification and isolation of stem cell populations, particularly hematopoietic stem cells

• Characterization of side-population cells based on dye efflux properties

• Studying multidrug resistance mechanisms in cancer cells

• Investigating ABCG2's role in protecting stem cells through its anti-apoptotic effects

According to published research using these antibodies, flow cytometry applications have been documented in multiple studies examining ABCG2 expression in human cells, particularly in cancer research and stem cell biology .

What are the optimal storage conditions for ABCG2 antibodies?

For maintaining optimal activity of ABCG2 antibodies, particularly APC-conjugated versions, the following storage conditions are recommended:

• Store at 2 to 8°C for up to 12 months from date of receipt as supplied

• Protect from light due to the photosensitive nature of the APC fluorophore

• Do not freeze as this can damage the protein structure and fluorescent conjugate

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation

These conditions are essential for preserving antibody integrity and fluorescent signal strength when used in applications like flow cytometry.

How should researchers optimize ABCG2 antibody staining for flow cytometry?

When using ABCG2 antibodies (such as Clone 5D3) for flow cytometry, researchers should:

• Determine optimal antibody dilutions empirically for each cell type and application

• Include appropriate isotype controls (such as IC0041A when using FAB995A) to establish background staining levels

• Follow established protocols for staining membrane-associated proteins

• When analyzing results, use filled histograms for the ABCG2 antibody staining and open histograms for isotype controls to clearly visualize specific binding

• For MCF-7 cells specifically, a distinct positive population should be observable when compared to the isotype control

For detecting rare stem cell populations, consider enriching for lineage-negative cells before staining to increase detection sensitivity. Additional markers may be needed to fully characterize side-population cells expressing ABCG2.

What controls should be included when characterizing ABCG2+ cell populations?

A comprehensive control strategy for ABCG2 antibody experiments includes:

Researchers should additionally validate their findings using secondary methods such as Western blotting or qPCR to confirm protein expression levels detected by flow cytometry .

How can ABCG2 antibodies be used to study cancer stem cells and drug resistance?

ABCG2 antibodies serve as powerful tools for investigating cancer stem cells (CSCs) and drug resistance mechanisms through several approaches:

• Identification and isolation of CSCs: Studies like Zhao et al. (2013) demonstrated that ABCG2 antibodies can identify side populations in esophageal cancer that exhibit stem cell-like properties and contribute to chemotherapy resistance and metastasis .

• Correlation with treatment outcomes: Gojo et al. (2013) used ABCG2 antibodies to examine transporter expression in acute myeloid leukemia patients before and after treatment with vorinostat combined with cytarabine and etoposide, providing insights into treatment resistance mechanisms .

• Characterization of multidrug resistance: Bram et al. (2007) utilized ABCG2 antibodies to investigate how C421 allele-specific ABCG2 gene amplification confers resistance to antitumor compounds in human lung cancer cells .

• Isolation of melanoma stem cells: Keshet et al. (2008) employed flow cytometry with transporter antibodies to identify and characterize melanoma stem cells based on expression of drug resistance transporters .

These applications highlight how ABCG2 antibodies contribute to understanding the relationship between stem cell properties and therapeutic resistance in cancer.

What insights can conformational-specific ABCG2 antibodies provide about transporter function?

Conformational-specific antibodies like clone 5D3 offer unique insights into ABCG2 structure and function:

• Ozvegy-Laczka et al. (2008) demonstrated that interaction between ABCG2 and the 5D3 monoclonal antibody is regulated by intramolecular rearrangements rather than covalent dimer formation . This research revealed that:

  • 5D3 antibody binding is sensitive to the conformational state of ABCG2

  • ATP binding and hydrolysis induce conformational changes that alter antibody recognition

  • These conformational shifts occur independent of transporter dimerization

  • Specific residues in the extracellular loops contribute to the 5D3 epitope

• This ability to detect conformational states provides researchers with:

  • A tool to study the mechanism of substrate transport

  • Methods to assess inhibitor effects on transporter conformation

  • Means to investigate how mutations impact protein structure

  • Approaches to evaluate the functional status of ABCG2 in live cells

These conformational insights are particularly valuable for drug development and understanding how ABCG2 contributes to multidrug resistance phenotypes in cancer.

How do genomic variations in ABCG2 affect antibody binding and experimental interpretation?

Genetic variations in ABCG2 can significantly impact antibody binding and experimental outcomes:

• Allelic variants affect binding: Studies like Bram et al. (2007) demonstrated that the C421 allele-specific ABCG2 gene amplification not only confers drug resistance but can also alter the binding characteristics of antibodies .

• Expression level variations: ABCG2 expression appears greatest on CD34- cells, with variations across different stem cell populations. These differences must be considered when interpreting antibody staining patterns .

• Conformational effects of mutations: As shown by Ozvegy-Laczka et al. (2008), mutations can alter the conformational state of ABCG2, affecting recognition by conformation-sensitive antibodies like 5D3 .

• Single nucleotide polymorphisms (SNPs): Common SNPs in ABCG2 can modify protein folding, membrane localization, and function, potentially creating false negatives in antibody-based detection systems.

Researchers should consider sequencing the ABCG2 gene in their experimental model systems to account for these variations when interpreting antibody binding results, particularly when comparing data across different cell lines or patient samples.

What are common pitfalls when using ABCG2 antibodies in flow cytometry and how can they be addressed?

When using ABCG2 antibodies in flow cytometry, researchers frequently encounter these challenges:

• Low detection sensitivity:

  • Solution: Optimize staining conditions with titration experiments

  • Use brightness-enhanced fluorophores like APC for better signal-to-noise ratio

  • Consider signal amplification systems for very low expression levels

• High background staining:

  • Solution: Include proper blocking steps (Fc block, serum)

  • Ensure strict adherence to wash protocols

  • Always use appropriate isotype controls (e.g., IC0041A for FAB995A)

• Inconsistent results between experiments:

  • Solution: Standardize cell preparation procedures

  • Use calibration beads to normalize fluorescence intensity

  • Maintain consistent antibody lot numbers when possible

• False negative results:

  • Solution: Verify ABCG2 function using dye efflux assays

  • Include positive control cell lines (e.g., MCF-7)

  • Consider using multiple antibody clones targeting different epitopes

• Interference from dead/dying cells:

  • Solution: Include viability dye to exclude non-viable cells

  • Optimize sample preparation to maximize cell viability

  • Process samples quickly after collection

Addressing these issues systematically will improve data reliability and reproducibility when working with ABCG2 antibodies.

How can researchers validate ABCG2 antibody specificity in their experimental systems?

A comprehensive validation strategy for ABCG2 antibodies should include:

• Multiple detection methods:

  • Compare flow cytometry results with Western blot, immunohistochemistry, or immunofluorescence

  • Verify protein expression with mRNA quantification (RT-qPCR)

  • Use mass spectrometry for definitive protein identification when possible

• Genetic approaches:

  • Test antibody binding in ABCG2 knockout models

  • Use ABCG2 overexpression systems as positive controls

  • Employ siRNA/shRNA knockdown to create specificity controls

• Functional correlation:

  • Compare antibody binding with functional assays (e.g., Hoechst 33342 efflux)

  • Use ABCG2 inhibitors to block function and observe antibody binding changes

  • Assess correlation between staining intensity and functional capacity

• Cross-reactivity assessment:

  • Test against related ABC transporters (ABCB1, ABCC1)

  • Evaluate binding in species cross-reactivity panels

  • Examine binding to transfected cells expressing only the target protein

These validation steps ensure that observed signals truly represent ABCG2 expression and not artifacts or cross-reactivity with other proteins.

How are ABCG2 antibodies being used in single-cell analysis technologies?

ABCG2 antibodies are increasingly incorporated into cutting-edge single-cell analysis platforms:

• Single-cell RNA sequencing (scRNA-seq) with protein detection:

  • ABCG2 antibodies conjugated to oligonucleotide barcodes enable simultaneous detection of surface protein and transcriptome

  • This approach reveals relationships between ABCG2 protein expression and gene expression programs at single-cell resolution

• Mass cytometry (CyTOF):

  • Metal-conjugated ABCG2 antibodies allow multiplexed phenotyping of rare stem cell populations

  • Enables characterization of ABCG2+ cells in complex tissues without fluorescence spillover constraints

• Circulating tumor cell (CTC) analysis:

  • ABCG2 antibodies help identify stem-like CTCs in cancer patients

  • Gallerani et al. (2021) used single CTC profiling in esophageal cancer patients during therapy to reveal unexplored molecular pathways

• Spatial transcriptomics:

  • Combining ABCG2 antibody staining with spatial transcriptomics reveals the distribution and molecular characteristics of ABCG2+ cells within their tissue microenvironment

These emerging applications provide unprecedented insights into the heterogeneity and functional significance of ABCG2-expressing cells in both normal tissues and disease states.

What is the relationship between ABCG2 expression and immunotherapy response?

Emerging research suggests important connections between ABCG2 expression and immunotherapy outcomes:

• Immune evasion mechanisms:

  • ABCG2-expressing cancer stem cells may exhibit altered antigen presentation

  • These cells often show reduced expression of stress ligands recognized by NK cells

  • ABCG2 transporters can modulate the tumor microenvironment through efflux of small molecules that affect immune cell function

• Impact on checkpoint inhibitor therapy:

  • ABCG2+ tumor cells may respond differently to checkpoint blockade

  • Some checkpoint inhibitor drugs may be substrates for ABCG2, affecting local drug concentrations

  • ABCG2 expression correlates with immunosuppressive phenotypes in some cancer types

• Combination therapy considerations:

  • ABCG2 inhibitors could potentially sensitize resistant cell populations to immunotherapy

  • Sequential therapy targeting ABCG2+ cells before immunotherapy might improve outcomes

  • Monitoring ABCG2+ populations during treatment could provide biomarkers for response prediction

Researchers can use ABCG2 antibodies to investigate these relationships through:

• Multiparameter flow cytometry combining immune and stem cell markers

• Analysis of pre- and post-treatment biopsies to track ABCG2+ population dynamics

• Correlation of ABCG2 expression with immunotherapy response in clinical samples

This emerging field offers opportunities to improve immunotherapy strategies through better understanding of ABCG2+ cell populations.