HUS1B Antibody

What is HUS1B and how does it differ from HUS1?

HUS1B is most closely related to HUS1, a component of a cell cycle checkpoint protein complex involved in cell cycle arrest in response to DNA damage. While sharing structural similarities, HUS1B exhibits distinct functional characteristics. Unlike HUS1, HUS1B can interact with the checkpoint protein RAD1 but not with RAD9. Notably, overexpression of HUS1B has been shown to induce cell death, suggesting a related but functionally distinct role compared to HUS1 .

HUS1B has the following molecular identifiers:

• UniProt ID: Q8NHY5 (Human)

• Entrez Gene ID: 135458 (Human), 691382 (Rat)

• Protein aliases include: Checkpoint protein HUS1B, hHUS1B, HUS1 checkpoint homolog b, MGC126746, MGC126748, RP11-532F6.1

What are the primary applications for HUS1B antibodies in research?

HUS1B antibodies are primarily utilized in Western Blot (WB) and ELISA applications in research settings . When comparing to the related HUS1 antibodies, which have broader application potential including Immunohistochemistry (IHC) and Immunofluorescence (IF), researchers should note that HUS1B antibodies currently have more limited validated applications . This difference in application range reflects both the distinct properties of these proteins and the current state of antibody development for HUS1B.

The evidenced applications for HUS1B antibodies include:

What is known about HUS1B expression patterns in various cell types?

HUS1B expression shows a distinctive pattern across tissues and cell types, though less extensively characterized than HUS1. According to the Pharos database, HUS1B has a knowledge value of 0.56 (on a 0-1 scale) regarding cell type or tissue expression patterns . This indicates moderate but not comprehensive knowledge of its expression profile.

Key knowledge metrics for HUS1B include:

When designing experiments, researchers should consult resources such as The Human Protein Atlas to identify appropriate positive control cell lines that express HUS1B . This step is crucial for establishing experimental validity before proceeding with studies on your cell line of interest.

How should I design a flow cytometry experiment to detect HUS1B?

When designing a flow cytometry experiment to detect HUS1B, researchers must first determine the subcellular localization of HUS1B to select appropriate cell preparation methods. As HUS1B is primarily involved in DNA damage response pathways, it is likely to be predominantly intracellular.

The experimental design should include:

• Cell preparation: For intracellular proteins, fixation and permeabilization are required. Use a fixative compatible with your antibody (typically 4% paraformaldehyde) followed by a permeabilization agent appropriate for nuclear proteins .

• Controls:

  • Positive control: Cell line known to express HUS1B (consult Human Protein Atlas)

  • Negative control: Cell line with low/no HUS1B expression or HUS1B knockdown cells

  • Isotype control: Using appropriate isotype antibody to determine non-specific binding

  • Unstained control: For autofluorescence baseline

• Antibody selection and validation:

  • Confirm the clonality (polyclonal HUS1B antibodies are available)

  • Verify host species (rabbit IgG is common for available HUS1B antibodies)

  • Check species reactivity (human and rat for many commercially available antibodies)

  • Determine if direct or indirect detection will be used

• Protocol optimization:

  • Titrate antibody concentration (starting with manufacturer recommendations)

  • Optimize fixation and permeabilization conditions

  • Adjust incubation times and temperatures

Remember that understanding your antibody's specifics is crucial - knowing whether it targets intracellular or extracellular epitopes determines your permeabilization approach .

What are the optimal storage and handling conditions for HUS1B antibodies?

Proper storage and handling of HUS1B antibodies are crucial for maintaining their activity and specificity. Based on manufacturer recommendations, the following conditions should be observed:

What dilutions and conditions are recommended for Western blot applications with HUS1B antibodies?

For Western blot applications using HUS1B antibodies, researchers should consider the following recommended dilutions and conditions:

• Antibody dilution range: While specific HUS1B antibody dilution ranges weren't provided in the search results, comparable antibodies like HUS1 are typically used at dilutions of 1:300-1:1000 for Western blotting . As a starting point for HUS1B antibodies, a similar range can be tested, with optimization for your specific experimental conditions.

• Sample preparation considerations:

  • Ensure complete protein denaturation if using a denaturing gel system

  • Include appropriate protease inhibitors in lysis buffers

  • Consider phosphatase inhibitors if studying phosphorylation status

• Controls to include:

  • Positive control: Lysate from cells known to express HUS1B

  • Negative control: Lysate from cells with minimal HUS1B expression or HUS1B knockdown

  • Loading control: Housekeeping protein to ensure equal loading

• Expected molecular weight: Be aware that HUS1B may be observed at a molecular weight close to the related HUS1 protein, which has a calculated molecular weight of 32 kDa and is observed at 32 kDa in Western blots .

• Optimization tip: It is recommended that HUS1B antibodies should be titrated in each testing system to obtain optimal results, as the optimal dilution can be sample-dependent .

When detecting faint signals, researchers might consider using more sensitive detection systems or signal enhancement methods to improve visualization of low-abundance proteins like HUS1B.

How do I troubleshoot non-specific binding when using HUS1B antibodies?

Non-specific binding is a common issue when working with antibodies that can complicate data interpretation, especially with less well-characterized targets like HUS1B. Here are methodological approaches to identify and resolve non-specific binding:

• Validation of observed bands/signals:

  • Compare observed molecular weight with the expected 32 kDa (based on HUS1 data)

  • Utilize positive and negative control samples

  • Consider using different antibody clones targeting different epitopes of HUS1B

  • Implement knockdown or knockout controls where HUS1B expression is reduced/eliminated

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Antibody incubation adjustments:

  • Reduce primary antibody concentration

  • Optimize incubation temperature (4°C overnight vs. room temperature)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Consider adding low concentrations (0.1-0.5%) of non-homologous serum from the same species as your samples

• Washing optimization:

  • Increase the number of washes

  • Extend washing duration

  • Use buffers with higher salt concentration or detergent content

• Cross-reactivity assessment:

  • Test the antibody on samples known to lack HUS1B

  • Perform peptide competition assays to confirm specificity

  • Evaluate potential cross-reactivity with HUS1 and other related proteins

When analyzing results, carefully examine background levels and compare signal-to-noise ratios across different optimization conditions to determine the most specific detection parameters.

How can I differentiate between HUS1 and HUS1B signals in my experiments?

Differentiating between HUS1 and HUS1B signals is critical for accurate data interpretation, given their sequence similarities and related functions. Here's a methodological approach to ensure signal specificity:

• Antibody selection strategy:

  • Choose antibodies raised against non-conserved regions between HUS1 and HUS1B

  • Verify antibody specificity through manufacturer validation data

  • Consider using antibodies that have been validated in knockout/knockdown systems

• Experimental controls:

  • Include HUS1 knockout/knockdown samples to confirm HUS1B signal specificity

  • Similarly, include HUS1B knockout/knockdown samples to confirm HUS1 signal specificity

  • Use cell lines with differential expression of HUS1 versus HUS1B

• Analytical approaches:

  • Perform immunoprecipitation followed by mass spectrometry to confirm protein identity

  • Use dual staining with antibodies targeting different epitopes on each protein

  • Consider differences in molecular interactions (HUS1B interacts with RAD1 but not RAD9, unlike HUS1)

• Functional discrimination:

  • Design experiments leveraging the unique cell death induction property of HUS1B overexpression

  • Utilize co-immunoprecipitation with RAD1 (which interacts with HUS1B) versus RAD9 (which interacts with HUS1 but not HUS1B)

When analyzing Western blot data, note that while both proteins have similar molecular weights, slight differences in migration patterns might be observable under high-resolution gel conditions. Always validate findings through multiple experimental approaches to confirm the identity of detected proteins.

What are the common pitfalls when interpreting flow cytometry data for HUS1B detection?

When interpreting flow cytometry data for HUS1B detection, researchers should be aware of several common pitfalls that can lead to misinterpretation:

• Autofluorescence interference:

  • Certain cell types exhibit high autofluorescence that can mask true signals

  • Always include unstained controls for each cell type

  • Consider using spectral compensation to correct for autofluorescence

• Fixation and permeabilization artifacts:

  • Overaggressive permeabilization can destroy epitopes or create non-specific binding

  • Insufficient permeabilization may prevent antibody access to intracellular HUS1B

  • Different fixatives can affect antibody binding differently; validate fixation protocols

• Population gating challenges:

  • Inappropriate gating can exclude relevant cell populations or include debris

  • Use forward/side scatter profiles to exclude cell debris and doublets

  • Include viability dyes to exclude dead cells that often show non-specific binding

• Signal intensity interpretation:

  • Low signal-to-noise ratio can make it difficult to distinguish positive from negative populations

  • Use isotype controls to establish background staining levels

  • Be cautious about interpreting small shifts in fluorescence as positive signals

• Control-related issues:

  • Failure to include proper positive and negative controls

  • Not validating antibody specificity in the context of flow cytometry

  • Using controls processed differently than experimental samples

• Cell cycle effects:

  • As HUS1B is involved in cell cycle regulation, its expression may vary with cell cycle phase

  • Consider including cell cycle phase markers for more nuanced interpretation

  • Compare populations at similar cell cycle stages for more accurate comparisons

To improve data reliability, quantify signal-to-noise ratios, perform replicate experiments, and validate findings using complementary techniques such as Western blotting or immunofluorescence microscopy.

How can I use HUS1B antibodies to study DNA damage response pathways?

HUS1B antibodies can be powerful tools for investigating DNA damage response (DDR) pathways, given HUS1B's role in cell cycle checkpoint regulation. Here's a methodological approach for such studies:

• Experimental design for DNA damage induction:

  • Use genotoxic agents (e.g., UV radiation, cisplatin, or hydroxyurea) to induce specific types of DNA damage

  • Apply time-course experiments to track HUS1B protein levels and localization following damage

  • Compare HUS1B responses to those of the better-characterized HUS1 protein

• Co-immunoprecipitation studies:

  • Use HUS1B antibodies to pull down protein complexes

  • Analyze interacting partners, particularly focusing on RAD1 interactions

  • Compare interaction profiles between damaged and undamaged conditions

• Chromatin association studies:

  • Employ chromatin fractionation followed by Western blotting with HUS1B antibodies

  • Track HUS1B recruitment to chromatin following various types of DNA damage

  • Correlate chromatin association with cell cycle phases

• Knockdown/knockout complementation:

  • In HUS1B-depleted cells, study the rescue effects of wild-type versus mutant HUS1B

  • Use HUS1B antibodies to confirm expression levels of exogenous proteins

  • Analyze checkpoint activation and cell survival outcomes

• Comparative analysis with HUS1:

  • Given that HUS1B and HUS1 likely have distinct roles, use antibodies against both proteins to compare:

    • Temporal activation patterns

    • Subcellular localization changes

    • Protein stability following DNA damage

    • Interactions with other checkpoint proteins

• Cell death pathway investigation:

  • Leverage HUS1B's unique property of inducing cell death when overexpressed

  • Use HUS1B antibodies to correlate expression levels with apoptotic markers

  • Investigate whether HUS1B-induced cell death occurs through classical apoptosis or alternative pathways

When designing these experiments, ensure proper controls are in place, including both positive controls (known DDR proteins like γH2AX) and negative controls (non-DDR proteins) to validate your findings.

What approaches can be used to study HUS1B phosphorylation status and its functional implications?

Studying protein phosphorylation is critical for understanding signaling cascades, particularly in DNA damage response pathways where HUS1B operates. Here's a methodological framework to investigate HUS1B phosphorylation:

• Phosphorylation site prediction and analysis:

  • Utilize bioinformatics tools (NetPhos, PhosphoSitePlus) to predict potential phosphorylation sites on HUS1B

  • Compare conservation of these sites with the related HUS1 protein

  • Focus on kinase motifs relevant to DNA damage response (ATM/ATR, CHK1/CHK2)

• Phosphorylation-specific detection methods:

  • Phospho-specific antibodies: If available, use antibodies recognizing specific phosphorylated residues

  • Phos-tag SDS-PAGE: Use this specialized gel system to separate phosphorylated from non-phosphorylated forms

  • Mass spectrometry: For unbiased identification of phosphorylation sites and stoichiometry

• Phosphorylation induction and temporal dynamics:

  • Treat cells with DNA damaging agents and analyze HUS1B phosphorylation status over time

  • Use kinase inhibitors to identify responsible kinases

  • Compare phosphorylation patterns across different damage types (UV, IR, replication stress)

• Functional significance assessment:

  • Generate phospho-mimetic (S/T→D/E) and phospho-deficient (S/T→A) mutants

  • Perform rescue experiments in HUS1B-depleted cells

  • Analyze effects on:

    • Protein-protein interactions (particularly with RAD1)

    • Chromatin association

    • Cell cycle checkpoint activation

    • Cell death induction

• Comparative studies with HUS1:

  • Determine whether HUS1B shares phosphorylation sites with HUS1

  • Investigate whether the same kinases target both proteins

  • Analyze differences in phosphorylation-dependent functions

When conducting these experiments, researchers should be mindful that phosphorylation events may be transient and occur at low stoichiometry, requiring sensitive detection methods and careful optimization of experimental conditions.

How can HUS1B antibodies be applied in cancer research and potential therapeutic development?

Given HUS1B's involvement in cell cycle regulation and its ability to induce cell death when overexpressed, it represents a potential target for cancer research. Here's how HUS1B antibodies can be applied in oncology research contexts:

• Expression profiling across cancer types:

  • Use HUS1B antibodies for immunohistochemistry (IHC) analysis of tissue microarrays

  • Compare HUS1B expression between normal and tumor tissues

  • Correlate expression levels with clinical outcomes and tumor characteristics

  • Consider using the same approach as validated for HUS1 antibodies, which have established IHC protocols

• Functional studies in cancer models:

  • Investigate the consequences of HUS1B modulation (overexpression/knockdown) in cancer cell lines

  • Use HUS1B antibodies to confirm manipulation efficiency

  • Monitor effects on:

    • Cell proliferation

    • Apoptosis resistance

    • DNA damage response efficiency

    • Chemotherapy/radiotherapy sensitivity

• Mechanism of HUS1B-induced cell death:

  • Leverage the observation that HUS1B overexpression induces cell death

  • Characterize the cell death mechanism (apoptosis, necrosis, other)

  • Determine whether this mechanism is enhanced or suppressed in cancer cells

  • Investigate potential for therapeutic exploitation

• Synthetic lethality screening:

  • Identify drugs or genetic alterations that create synthetic lethality with HUS1B status

  • Use HUS1B antibodies to stratify cell lines or patient samples by expression level

  • Test whether HUS1B expression predicts sensitivity to DNA-damaging agents or checkpoint inhibitors

• Biomarker potential assessment:

  • Evaluate HUS1B as a potential diagnostic or prognostic biomarker

  • Develop standardized protocols for HUS1B detection in clinical samples

  • Correlate HUS1B with established biomarkers of DNA damage response

• Drug development applications:

  • Use HUS1B antibodies in target engagement studies for drug candidates

  • Screen for compounds that modulate HUS1B expression or function

  • Monitor HUS1B as a pharmacodynamic marker in treatment response

When conducting these studies, it's essential to validate findings across multiple cancer models and correlate experimental results with clinical data whenever possible to establish translational relevance.

How can single-cell analysis techniques be applied to study HUS1B expression and function?

Single-cell analysis technologies offer unprecedented insights into cellular heterogeneity and can be valuable for studying HUS1B in complex biological contexts:

• Single-cell RNA sequencing (scRNA-seq) applications:

  • Profile HUS1B transcript levels across individual cells in heterogeneous populations

  • Correlate HUS1B expression with cell cycle phase markers

  • Identify cell subpopulations with distinctive HUS1B expression patterns

  • Analyze co-expression patterns with other DNA damage response genes

• Single-cell protein analysis methods:

  • Mass cytometry (CyTOF): Incorporate HUS1B antibodies into CyTOF panels to simultaneously measure multiple proteins

  • Single-cell Western blotting: Quantify HUS1B protein levels in individual cells

  • Imaging flow cytometry: Combine flow cytometry with microscopy to correlate HUS1B levels with subcellular localization

• Spatial transcriptomics and proteomics:

  • Visualize HUS1B expression patterns within tissue architecture

  • Correlate HUS1B distribution with microenvironmental features

  • Examine HUS1B expression in relation to tissue pathology

• Integrative multi-omics approaches:

  • Combine single-cell transcriptomics, proteomics, and epigenomics

  • Create comprehensive maps of HUS1B regulation and function

  • Identify cell state-specific roles of HUS1B

• Live-cell analysis of HUS1B dynamics:

  • Use antibody fragments or nanobodies for live-cell imaging

  • Track HUS1B protein dynamics in response to DNA damage

  • Monitor interactions with partners like RAD1 using proximity ligation techniques

When implementing these advanced approaches, researchers should be mindful of technical challenges, including antibody specificity validation in each new platform, potential fixation artifacts, and the need for computational expertise to analyze complex datasets.

What are the considerations for developing multiplexed assays that include HUS1B detection?

Developing multiplexed assays that incorporate HUS1B detection allows researchers to study this protein in the context of broader signaling networks. Here are methodological considerations for successful multiplexing:

• Antibody panel design principles:

  • Antibody clone selection: Choose HUS1B antibodies with minimal cross-reactivity

  • Host species diversification: Select antibodies from different host species to enable simultaneous detection

  • Fluorophore/reporter selection: Consider spectral overlap and signal strength when choosing reporters

  • Epitope accessibility: Ensure fixation and permeabilization conditions preserve all target epitopes

• Validation requirements for multiplexed assays:

  • Single-stain controls: Test each antibody individually before combining

  • Fluorescence minus one (FMO) controls: Essential for setting accurate gates

  • Blocking optimization: Prevent non-specific binding in complex antibody mixtures

  • Signal-to-noise optimization: Adjust antibody concentrations to achieve balanced signal intensities

• Platform-specific considerations:

  • Flow cytometry: Compensation and panel design to minimize spectral overlap

  • Mass cytometry: Metal-conjugated antibodies require validation for sensitivity and specificity

  • Multiplexed immunofluorescence: Sequential staining may be needed to avoid host species conflicts

  • Multiplexed Western blotting: Consider protein size to avoid signal overlap

• Data analysis approaches:

  • Dimensionality reduction techniques (tSNE, UMAP) for visualizing high-parameter data

  • Clustering algorithms to identify cell populations with distinct patterns

  • Correlation analyses between HUS1B and other markers

  • Machine learning classification of cell states based on marker patterns

• Biological context optimization:

  • Include cell cycle markers to contextualize HUS1B signals

  • Add DNA damage markers to correlate with HUS1B function

  • Consider including HUS1 detection for comparative analysis

  • Include markers for cell death pathways given HUS1B's role in cell death induction

When implementing multiplexed assays, thorough validation of each component and careful optimization of staining protocols are essential to ensure reliable, interpretable results.

Future research directions and emerging technologies

The field of HUS1B research stands to benefit significantly from emerging technologies and methodological advances. Based on current knowledge gaps and technological trends, several promising research directions include:

• Enhanced antibody development: Creation of more specific, sensitive antibodies against HUS1B, including phospho-specific antibodies to study post-translational modifications.

• Application of proximity-based techniques: Implementation of proximity ligation assays and BioID approaches to better characterize HUS1B protein interactions in situ.

• CRISPR-based functional genomics: Systematic analysis of HUS1B function through CRISPR knockout, knockin, and base editing technologies.

• Single-cell and spatial biology approaches: Integration of HUS1B analysis in single-cell multi-omics and spatial transcriptomics/proteomics to understand context-dependent functions.

• Translational research applications: Investigation of HUS1B as a potential biomarker or therapeutic target, particularly given its role in cell death induction and potential implications for cancer treatment resistance .