ARL2BP Antibody, HRP conjugated

What is ARL2BP and what is its biological function?

ARL2BP (ADP-ribosylation factor-like protein 2-binding protein) functions primarily as a binding partner of ARL2. Together with ARL2, it plays a significant role in the nuclear translocation, retention, and transcriptional activity of STAT3 . The protein may also serve as an effector of ARL2 in various cellular pathways . ARL2BP is also known by several alternative names including BART, BART1, ARF-like 2-binding protein, and Binder of ARF2 protein 1 .

What are the key characteristics of HRP-conjugated ARL2BP antibodies?

HRP-conjugated ARL2BP antibodies are immunological reagents with horseradish peroxidase directly linked to the antibody molecule, enabling direct detection without secondary antibodies. These antibodies are typically available in polyclonal formats raised in rabbits . The immunogen commonly used is recombinant human ARL2BP (amino acids 1-163), and the antibodies are purified via antigen affinity chromatography . They are preserved in buffers containing 0.01M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol, and should be stored at -20°C or -80°C to maintain reactivity .

How can knockout validation be used to confirm ARL2BP antibody specificity?

Knockout validation represents a gold standard approach for confirming antibody specificity. For ARL2BP antibodies, specificity has been demonstrated using ARL2BP knockout HeLa cell lines (ab265269) . In these experiments, antibodies show clear signals in wild-type HeLa cells but complete signal loss in the knockout cells when analyzed by Western blot . This validation methodology employs simultaneous detection of loading controls (typically GAPDH at 36 kDa) to confirm equal protein loading across samples . Researchers should consider including similar knockout controls in their experimental design to definitively establish antibody specificity.

What are the optimal antigen retrieval conditions for ARL2BP detection in FFPE tissues?

For optimal detection of ARL2BP in formalin-fixed paraffin-embedded (FFPE) tissues, heat-mediated antigen retrieval using Tris/EDTA buffer at pH 9.0 is recommended before commencing immunohistochemical staining protocols . This specific buffer system has been validated on multiple tissue types including human kidney and rat liver samples . Alternative antigen retrieval may be performed with citrate buffer at pH 6.0, but effectiveness may vary depending on tissue type and fixation conditions . Research indicates that insufficient antigen retrieval represents a primary cause of false-negative results in ARL2BP immunohistochemistry.

How does ARL2BP subcellular localization inform experimental design for immunofluorescence studies?

ARL2BP exhibits specific subcellular localization patterns that should inform immunofluorescence experimental design. In A549 cells, ARL2BP can be detected following 4% paraformaldehyde fixation at dilutions of approximately 1/100, followed by fluorophore-conjugated secondary antibodies . The protein demonstrates distinct localization patterns that correlate with its functional role in STAT3 nuclear translocation . Researchers should employ nuclear counterstains such as DAPI to properly contextualize ARL2BP distribution patterns and consider co-localization studies with STAT3 or ARL2 to further elucidate functional relationships.

What cross-reactivity considerations exist when using ARL2BP antibodies across different species?

Species cross-reactivity is an important consideration when selecting ARL2BP antibodies. While HRP-conjugated polyclonal antibodies are primarily validated for human ARL2BP detection , non-conjugated monoclonal antibodies demonstrate broader cross-reactivity profiles encompassing human, mouse, and rat samples . This cross-reactivity has been validated in multiple experimental systems including Western blot analysis of mouse brain, heart, and rat spleen tissue lysates . Researchers working with non-human models should carefully examine sequence homology and validation data before selecting an appropriate antibody.

How can non-specific binding be minimized when using HRP-conjugated ARL2BP antibodies?

Non-specific binding with HRP-conjugated antibodies can be minimized through several methodological approaches. First, optimize blocking conditions using 5% BSA or 5% non-fat milk in TBS-T for Western applications . Second, determine the optimal antibody dilution through titration experiments; for HRP-conjugated ARL2BP antibodies, starting dilutions for ELISA applications should be empirically determined for each experimental system . Third, include appropriate negative controls such as isotype-matched irrelevant antibodies and/or knockout/knockdown samples . Finally, consider including competing peptides when available to demonstrate binding specificity.

What are the recommended protocols for immunoprecipitation of ARL2BP?

For successful immunoprecipitation of ARL2BP, researchers should follow validated protocols that have demonstrated effective protein pulldown. Non-conjugated ARL2BP antibodies have been successfully used for immunoprecipitation from HepG2 cell lysates at a dilution of 1/50 . The immunoprecipitation should include appropriate controls (e.g., PBS instead of cell lysate) and detection via Western blot using the same or different ARL2BP antibodies . For optimal results, pre-clearing lysates with protein A/G beads before antibody addition can reduce non-specific binding, and gentle washing conditions help maintain protein complex integrity.

How should freeze-thaw cycles be managed for HRP-conjugated antibodies?

HRP-conjugated antibodies, including ARL2BP antibodies, are particularly sensitive to repeated freeze-thaw cycles that can compromise both antibody binding capacity and enzymatic activity . To maintain optimal performance, aliquot antibodies upon receipt and store at -20°C or -80°C . Each aliquot should be sized appropriately for single experimental use to avoid repeated freezing and thawing. When removing from storage, thaw aliquots rapidly in a room temperature water bath but keep on ice after thawing. For longer-term storage at working dilution, 4°C is recommended with the addition of preservatives like sodium azide (note: azide inhibits HRP, so should not be used with working dilutions).

What is the recommended approach for validating ARL2BP antibodies in new experimental systems?

When adapting ARL2BP antibodies to new experimental systems, a structured validation approach is essential. First, perform Western blot analysis to confirm detection of the correct molecular weight band (19-20 kDa for ARL2BP) . Second, include positive controls such as A549 cells or human fetal brain lysate where ARL2BP expression has been confirmed . Third, implement negative controls through siRNA knockdown, CRISPR knockout, or tissues/cells known to lack ARL2BP expression . Fourth, consider orthogonal validation through mass spectrometry or recombinant protein spike-in experiments. Finally, validate specificity in the intended application (ELISA, IF, etc.) as antibody performance can vary between applications.

How do polyclonal and monoclonal ARL2BP antibodies compare in research applications?

Polyclonal and monoclonal ARL2BP antibodies exhibit distinct characteristics that influence their application suitability. Polyclonal antibodies, including HRP-conjugated versions, recognize multiple epitopes on the ARL2BP protein, potentially offering higher sensitivity but with greater batch-to-batch variation . Monoclonal antibodies, such as clone EPR15265, provide consistent epitope recognition with high specificity, making them particularly valuable for quantitative applications and longitudinal studies . Monoclonal antibodies have been extensively validated across multiple applications including Western blot, immunoprecipitation, immunofluorescence, flow cytometry, and immunohistochemistry . The choice between polyclonal and monoclonal formats should be guided by the specific experimental requirements, with monoclonals preferred for applications demanding high reproducibility.

What cell and tissue types have been validated for ARL2BP antibody applications?

ARL2BP antibodies have been validated across diverse cell and tissue types. In cell lines, successful detection has been demonstrated in HeLa, A549, HepG2, HUVEC, RAW 264.7, and NIH/3T3 cells . For tissue applications, validation has been performed in human kidney, rat liver, mouse brain, mouse heart, and rat spleen samples . This broad validation profile suggests robust applicability across different experimental systems. When working with novel cell or tissue types, researchers should start with validated sample types as positive controls before progressing to untested systems.

What are the optimal fixation conditions for ARL2BP detection in immunofluorescence?

For optimal detection of ARL2BP by immunofluorescence, 4% paraformaldehyde fixation has been successfully validated . This fixation protocol preserves both protein antigenicity and cellular architecture. Following fixation, permeabilization with 90% methanol allows antibody access to intracellular antigens when performing flow cytometry applications . For immunocytochemistry, alternative permeabilization agents such as 0.1-0.5% Triton X-100 may also be effective. Researchers should evaluate fixative impact on epitope accessibility for their specific experimental system, as overfixation can mask epitopes and reduce signal intensity.

How can flow cytometry protocols be optimized for intracellular ARL2BP detection?

Optimization of flow cytometry protocols for intracellular ARL2BP detection requires attention to several key parameters. First, effective cell fixation with 4% paraformaldehyde preserves cellular architecture while maintaining epitope accessibility . Second, permeabilization with 90% methanol is critical for antibody penetration to intracellular antigens . Third, antibody concentration should be carefully titrated; for non-conjugated antibodies, dilutions around 1/200 have proven effective for A549 cells . Fourth, appropriate negative controls including isotype-matched irrelevant antibodies (e.g., rabbit monoclonal IgG) should be included in each experiment . Finally, cell populations should be clearly defined using forward and side scatter parameters to eliminate debris and cell aggregates from analysis.

How does ARL2BP relate to STAT3 signaling pathways in experimental research?

ARL2BP, together with its binding partner ARL2, plays a critical role in STAT3 signaling by facilitating nuclear translocation, retention, and transcriptional activity of STAT3 . This functional relationship provides important research contexts for studying cancer biology, inflammation, and development. When designing experiments investigating this relationship, researchers should consider co-immunoprecipitation approaches to demonstrate physical interactions between ARL2BP and STAT3 components. Additionally, subcellular fractionation combined with Western blotting can provide evidence for ARL2BP's role in nuclear transport processes. Reporter gene assays measuring STAT3-dependent transcription represent another valuable approach for functional characterization of ARL2BP in this pathway.

What considerations should be made when using ARL2BP antibodies in multiplexed detection systems?

When incorporating ARL2BP antibodies into multiplexed detection systems, several technical considerations must be addressed. First, evaluate potential cross-reactivity between multiple primary antibodies, particularly when they originate from the same host species . Second, for HRP-conjugated antibodies, ensure compatible substrate systems that provide distinct visualization without signal overlap . Third, consider sequential rather than simultaneous detection if cross-reactivity or signal interference occurs. Fourth, when designing panels for flow cytometry or multiplex immunofluorescence, select fluorophores with minimal spectral overlap or implement appropriate compensation controls. Finally, validate the multiplexed system with single-stained controls to ensure antibody performance is not compromised in the multiplexed context.

How can quantitative analysis of ARL2BP expression be standardized across different experimental platforms?

Standardization of quantitative ARL2BP expression analysis requires implementation of several key practices. First, include recombinant ARL2BP protein standards at known concentrations to generate calibration curves for absolute quantification . Second, normalize ARL2BP signals to appropriate housekeeping proteins such as GAPDH for Western blot applications or to cell number/tissue area for imaging-based methods . Third, employ consistent antibody lots and dilutions across experimental series to minimize technical variation. Fourth, implement digital image analysis with standardized acquisition parameters and analysis algorithms. Finally, incorporate inter-laboratory validation samples when comparing data across different research sites to account for systematic variations in equipment and protocols.