ABCB9 Antibody

What is the function of ABCB9 protein and why is it important in research?

ABCB9 plays a crucial role in the immune response by facilitating peptide transport into the endoplasmic reticulum, where peptides can be loaded onto major histocompatibility complex (MHC) class I molecules for presentation to CD8+ T cells. This function is vital for recognizing intracellular pathogens and tumor cells, making ABCB9 an important player in the adaptive immune system .

ABCB9 forms homodimers and localizes in lysosomes, where it operates as an ATP-dependent peptide transporter with broad specificity for peptides ranging from 6 to 59 amino acids in length. Despite exhibiting low affinity for substrates, ABCB9 demonstrates high transport efficiency, underscoring its significance in cellular peptide processing and immune surveillance .

What applications are ABCB9 antibodies commonly used for?

ABCB9 antibodies are versatile tools suitable for multiple applications in molecular and cellular biology research. Based on the validated applications across multiple commercial sources, these antibodies can be used for:

These applications allow researchers to detect, localize, and quantify ABCB9 protein in various experimental settings .

What are the key considerations for selecting the appropriate ABCB9 antibody?

When selecting an ABCB9 antibody, researchers should consider:

• Specificity: Given documented issues with non-specific reactivity in certain ABCB9 antibodies, validate specificity using appropriate controls (e.g., knockout cells) .

• Species reactivity: Confirm that the antibody recognizes ABCB9 in your species of interest. Most commercial antibodies react with human and mouse ABCB9, with some also validated for rat .

• Application compatibility: Select antibodies validated for your specific application (WB, IHC, IF, etc.) .

• Clonality: Consider whether a monoclonal antibody (higher specificity) or polyclonal antibody (potentially higher sensitivity) better suits your experimental needs .

• Immunogen information: Understand what region of ABCB9 the antibody targets, as this may affect epitope accessibility in certain applications .

How should I optimize Western blot protocols for ABCB9 detection?

For optimal Western blot detection of ABCB9:

• Sample preparation: Use whole-cell lysates from tissues or cell lines known to express ABCB9 (e.g., HeLa, 293T, mouse NIH3T3, HL-60, K-562, BGC-823) .

• Loading amount: Load between 5-50 μg of protein lysate per lane; higher amounts may be needed for tissues with lower ABCB9 expression .

• Antibody concentration: Start with dilutions between 1:500-1:2000 for polyclonal antibodies or 0.04-1 μg/ml for monoclonal antibodies .

• Expected molecular weight: Look for a band between 72-84 kDa, though the calculated molecular weight is 84 kDa and observed weight may be 72 kDa .

• Exposure time: Begin with short exposures (3-10 seconds) when using chemiluminescence detection to avoid background signals .

• Controls: Include positive controls (cell lines known to express ABCB9) and negative controls where possible (knockout cells or tissues) .

What are the recommended protocols for immunoprecipitation of ABCB9?

For effective immunoprecipitation of ABCB9:

• Lysate preparation: Use 1.0-3.0 mg of total protein lysate per IP reaction .

• Antibody amount: Use 0.5-4.0 μg of ABCB9 antibody per IP reaction .

• Co-immunoprecipitation: When studying protein-protein interactions involving ABCB9 (e.g., with ABCB5β or ABCB6), immunoprecipitate with either anti-ABCB9, anti-ABCB5, or anti-ABCB6 antibody and detect the interacting partners by Western blotting using the corresponding antibodies .

• Controls: Include an isotype control to determine the specificity of the signals obtained in Western blots following IP .

• Sample sources: Mouse thymus tissue has been validated for successful IP of ABCB9 .

How should I optimize immunohistochemistry protocols for ABCB9 detection?

For optimal immunohistochemical detection of ABCB9:

• Tissue preparation: Use fresh frozen or formalin-fixed, paraffin-embedded tissues.

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results. Alternatively, citrate buffer pH 6.0 can be used, though results may vary .

• Antibody dilution: Start with dilutions between 1:50-1:500 .

• Detection system: Use an appropriate detection system compatible with the host species of the primary antibody (typically rabbit for most ABCB9 antibodies).

• Validated tissues: Mouse testis tissue has been validated for successful IHC detection of ABCB9 .

• Controls: Include positive control tissues known to express ABCB9 and negative controls (omitting primary antibody or using tissues from knockout animals) .

How can I validate the specificity of ABCB9 antibodies?

Research has shown that some commercial ABCB9 antibodies may exhibit non-specific reactivity. To validate specificity:

• Use knockout controls: The gold standard for antibody validation is testing on samples from ABCB9 knockout organisms. In a documented case, a commercial antibody recognized a protein with the expected molecular weight of ABCB9 even in TAP-L (ABCB9) knockout cells, indicating non-specific binding .

• Gene silencing: If knockout samples are unavailable, use RNAi-mediated silencing of ABCB9 (shRNA or siRNA) as an alternative approach to create negative control samples .

• Multiple antibodies: Use multiple antibodies targeting different epitopes of ABCB9 and compare results.

• Proximity ligation assay (PLA): This technique can confirm protein-protein interactions and antibody specificity simultaneously. In melanoma research, PLA confirmed interactions between ABCB5β-ABCB6 and ABCB5β-ABCB9 protein pairs. The specificity was demonstrated by the reduction in PLA signal after shRNA knockdown of ABCB6 or ABCB9 .

• PCR validation: When using genetic knockout models, verify the absence of the ABCB9 gene using multiple PCR tests targeting different regions of the gene .

What are common issues encountered with ABCB9 antibodies and how can they be addressed?

Common issues include:

• Cross-reactivity: Some ABCB9 antibodies may cross-react with unidentified proteins of similar molecular weight. Address this by using knockout cells or tissues as negative controls and verifying with alternative detection methods .

• Non-specific binding: When non-specific bands appear in Western blots, optimize blocking conditions, increase washing steps, and test different dilutions of the primary antibody.

• Variable results across applications: An antibody performing well in one application may not work in another. Verify that the antibody is validated for your specific application .

• Batch-to-batch variation: Even antibodies from the same supplier may show variation between batches. Consider testing new lots against your previously validated lot.

What controls should be included when working with ABCB9 antibodies?

To ensure reliable results:

• Positive controls: Include samples known to express ABCB9, such as:

  • Cell lines: HeLa, 293T, NIH3T3, HL-60, K-562, BGC-823

  • Tissues: Mouse thymus, human testis, mouse testis

• Negative controls:

  • Genetic: ABCB9/TAP-L knockout cells or tissues

  • Technical: Omit primary antibody while maintaining all other steps

  • RNAi: Cells treated with ABCB9-specific shRNA or siRNA

• Isotype controls: Include matched isotype antibodies to identify non-specific binding .

• Validation by multiple methods: Confirm findings using complementary techniques (e.g., IF and WB, or PLA and co-immunoprecipitation) .

How can I study heterodimeric complexes involving ABCB9?

Recent research has identified novel heterodimeric complexes involving ABCB9:

• NanoBRET assay: This technique can detect protein-protein interactions by measuring bioluminescence resonance energy transfer. Research has confirmed the formation of ABCB5β-ABCB9 heterodimers using this approach. For specificity validation, perform donor saturation assays where the amount of donor construct is held constant while increasing the acceptor construct .

• Co-immunoprecipitation: This classic approach can confirm heterodimer formation. In melanoma research, ABCB5β-ABCB9 interactions were confirmed by immunoprecipitating with either anti-ABCB5 or anti-ABCB9 antibodies and detecting the partner protein by Western blotting .

• Proximity Ligation Assay (PLA): This method visualizes protein interactions in situ. In melanoma cells, PLA confirmed ABCB5β-ABCB9 interactions, with specificity demonstrated by the reduction in signal after shRNA knockdown of ABCB9 .

• Expression systems: For functional studies, heterodimers can be expressed in insect cell expression systems. Recent research has successfully expressed ABCB5β-ABCB9 heterodimers in High-Five insect cells .

How can I investigate the functional role of ABCB9 in peptide transport and immune responses?

To study ABCB9's functional role:

• Transport assays: Measure ATP-dependent peptide transport in isolated membrane vesicles expressing ABCB9. Site-directed mutagenesis targeting the Walker B motif (replacing E with Q) can be used to abolish ATP hydrolysis as a negative control .

• Genetic manipulation: Use CRISPR-Cas9 or shRNA approaches to alter ABCB9 expression and assess the impact on peptide loading onto MHC class I molecules .

• Heterodimer functional analysis: For chimeric heterodimers like ABCB5β-ABCB9, prepare different mutants with mutations in the Walker B motif of either one or both interacting partners to dissect their respective contributions to transport activity .

• Immunological readouts: Assess changes in CD8+ T cell responses following manipulation of ABCB9 expression to understand its role in antigen presentation .

What are the latest research findings on ABCB9 structure and function in relation to disease?

Recent advances in ABCB9 research include:

• Novel heterodimeric complexes: The identification of ABCB5β-ABCB9 heterodimers in melanoma represents a significant advance in understanding ABCB9 biology. These heterodimers could potentially be involved in drug resistance mechanisms in cancer .

• Lysosomally-localized transport: ABCB9 operates as an ATP-dependent peptide transporter in lysosomes, with broad specificity for peptides ranging from 6 to 59 amino acids in length. This function may have implications for antigen processing and presentation .

• Immune surveillance: The role of ABCB9 in peptide transport makes it potentially important in immune recognition of cancer cells and intracellular pathogens .

• Transport efficiency: Despite exhibiting low affinity for substrates, ABCB9 demonstrates high transport efficiency, which may have implications for how it functions in cellular contexts and potential therapeutic targeting .

These findings open new avenues for investigating ABCB9's role in cancer biology, particularly in melanoma, and in immune responses to various diseases.