HTT4 Antibody

What is the HTT4/MW4-S antibody and what epitope does it recognize?

HTT4, formally designated as MW4-S, is a mouse monoclonal antibody developed by the Patterson lab in 2001 as part of a series of eight anti-HTT monoclonal antibodies (MW1-8) created for diagnostic and research applications in Huntington's disease studies . According to antibody validation databases, MW4-S recognizes a specific epitope within the Huntingtin protein. The antibody is available from the Developmental Studies Hybridoma Bank (DSHB) with catalog number MW4-S (lot #43251) and is registered in the Antibody Registry as AB_528293 . This antibody belongs to the mouse IgG class and is supplied at a concentration of approximately 0.02 μg/μl based on standardized characterization protocols .

What are the recommended applications for HTT4 antibody?

Based on vendor specifications and validation studies, MW4-S (HTT4) is recommended for multiple research applications including Western blot (Wb) and immunofluorescence (IF) . The antibody has been systematically validated for these applications using a standardized protocol comparing readouts between wild-type cells and HTT knockout cell lines, providing researchers with high confidence in antibody specificity . When planning experiments with HTT4 antibody, researchers should consider that cell lines with higher HTT expression levels, such as DMS 53 (which expresses HTT transcript at 6.1 log2 TPM+1), provide more robust detection signals compared to cell lines with lower expression levels .

How does HTT4 antibody compare to other anti-Huntingtin antibodies in Western blot experiments?

When comparing HTT4 (MW4-S) performance with other antibodies, researchers should note that it is one of twenty characterized Huntingtin antibodies tested under standardized conditions. Comprehensive performance comparison studies have been conducted using identical protocols across multiple antibodies, allowing for direct comparison . The relative performance of MW4-S in Western blot applications can be assessed against nineteen other antibodies including recombinant monoclonals (like EPR5526, EP867Y, D7F7), other mouse monoclonals from the MW series, and recombinant polyclonals . For Western blot applications specifically, antibodies should be selected based on their demonstrated specificity, signal-to-noise ratio, and reproducibility across experimental conditions.

What cell lines are most suitable for validating HTT4 antibody specificity?

The DMS 53 cell line has been identified as the most suitable cellular background for validating HTT4 antibody specificity due to its high endogenous expression of HTT transcripts (6.1 log2 TPM+1) . This cell line demonstrated superior detection compared to alternatives such as HAP1 (3.7 log2 TPM+1) and HEK293T. The following cell lines have been validated for HTT antibody testing:

For optimal results, researchers should validate HTT4 antibody in DMS 53 cells using both wild-type and HTT knockout lines to confirm specificity .

What is the MW series of Huntingtin antibodies and how does HTT4 fit within this collection?

The MW series represents a collection of eight monoclonal antibodies (MW1-MW8) developed by the Patterson laboratory in 2001 specifically targeting Huntingtin protein . HTT4 (MW4-S) is part of this well-characterized antibody panel designed for studying Huntington's disease. According to comprehensive validation data, the MW series antibodies recognize different epitopes of Huntingtin, making them valuable tools for different experimental applications . Within this collection, MW1-S and MW8-S are recommended for Western blot, immunoprecipitation, and immunofluorescence applications, while MW4-S along with MW3-S, MW5-S, MW6-S, and MW7-S are validated for Western blot and immunofluorescence but not specifically recommended for immunoprecipitation . This distinction is important when designing experimental workflows to investigate different aspects of Huntingtin biology.

How can HTT4 antibody be used to investigate Huntingtin's role in vesicular transport?

For investigating Huntingtin's role in vesicular transport, HTT4 antibody can be employed in sophisticated experimental designs based on Huntingtin's established function in facilitating dynein/dynactin-mediated vesicle transport . Research protocols can utilize HTT4 in immunofluorescence studies to visualize Huntingtin localization during vesicular trafficking events. Additionally, researchers can use HTT4 in combination with antibodies against dynein intermediate chain to investigate their colocalization, as Huntingtin has been demonstrated to interact directly with dynein intermediate chain through yeast two-hybrid and affinity chromatography assays .

For in vitro vesicular transport studies, protocols can be designed similar to those where anti-HTT antibodies were used to study vesicle motility along microtubules. In such experiments, HTT antibodies inhibited vesicular transport, suggesting Huntingtin's facilitating role in dynein-mediated vesicle motility . For mechanistic studies, researchers can combine HTT4 antibody with RNAi approaches targeting Huntingtin to observe Golgi disruption phenotypes similar to those resulting from compromised dynein/dynactin function .

What are the considerations when using HTT4 antibody for studying polyglutamine expansion mutations in Huntington's disease?

When investigating polyglutamine expansion mutations characteristic of Huntington's disease using HTT4 antibody, researchers must consider the epitope specificity of this antibody relative to the polyQ region. Given that the MW series antibodies recognize different epitopes of Huntingtin, researchers should verify whether HTT4 specifically recognizes regions affected by polyglutamine expansion . If studying mutant vs. wild-type Huntingtin, researchers may need to utilize multiple antibodies with different epitope specificities - for instance, combining HTT4 with other antibodies from the MW series that might have different sensitivities to the polyQ tract length.

Experimental design should include appropriate controls such as cells expressing wild-type and mutant Huntingtin with varying polyglutamine repeat lengths to assess potential differences in antibody affinity or binding patterns. For quantitative studies of mutant vs. wild-type protein levels, researchers should validate that HTT4 has comparable affinity for both forms of the protein to avoid misinterpretation of results .

How can HTT4 antibody be validated in knockout models to ensure specificity?

Rigorous validation of HTT4 antibody specificity requires implementation of a knockout validation strategy using isogenic cell line pairs. Following established protocols for antibody validation, researchers should:

• Generate HTT knockout cell lines in an appropriate background that expresses HTT at detectable levels (e.g., DMS 53 with HTT expression at 6.1 log2 TPM+1)

• Process wild-type and knockout samples in parallel using identical protocols

• Perform Western blot analysis to confirm presence of the expected band in wild-type cells and absence in knockout cells

• For immunofluorescence applications, compare staining patterns between wild-type and knockout cells, looking for specific signal reduction in knockout cells

This standardized approach has been successfully employed for validating multiple Huntingtin antibodies including HTT4 . The specificity validation should be performed for each experimental application (Western blot, immunofluorescence, or immunoprecipitation) as antibody performance may vary across applications. For optimal results, researchers should select cell lines based on transcriptomic databases like DepMap, focusing on those with HTT expression levels greater than 2.5 log2 (TPM+1) .

What are the technical considerations when using HTT4 antibody for immunoprecipitation studies?

• Optimization of antibody concentration: Begin with titration experiments to determine optimal antibody-to-protein ratios

• Pre-clearing of lysates: Implement pre-clearing steps to reduce non-specific binding

• Appropriate controls: Include isotype controls and ideally HTT knockout cell lysates as negative controls

• Validation of IP efficiency: Confirm pull-down efficiency by Western blot of input, flow-through, and eluted fractions

• Crosslinking considerations: For studying protein complexes, evaluate whether antibody crosslinking to beads improves results

For studying Huntingtin's interactions with partners like dynein/dynactin, researchers might consider alternative antibodies from the MW series that are explicitly validated for IP, such as MW1-S or MW8-S . These validated IP antibodies could provide more reliable results for protein-protein interaction studies.

How can HTT4 antibody be applied in studies investigating Huntingtin interactions with dynein/dynactin complexes?

HTT4 antibody can be strategically employed in multi-approach experimental designs investigating Huntingtin interactions with dynein/dynactin complexes. Based on established research, Huntingtin facilitates dynein/dynactin-mediated vesicle transport through direct interaction with dynein intermediate chain . To investigate these interactions, researchers can implement the following experimental approaches:

• Co-immunoprecipitation studies: Use HTT4 in combination with anti-dynein intermediate chain antibodies to confirm interaction in cellular contexts

• Proximity ligation assays: Apply HTT4 with anti-dynein antibodies to visualize and quantify protein-protein interactions in situ

• Vesicle motility assays: Utilize HTT4 to analyze how antibody binding affects vesicular transport along microtubules in reconstituted systems

• Competitive binding studies: Combine HTT4 with other antibodies targeting different Huntingtin domains to map interaction interfaces

Research has shown that anti-HTT antibodies can inhibit vesicular transport along microtubules, suggesting Huntingtin's facilitating role in dynein-mediated vesicle motility . Additionally, inhibiting dynein function in vivo results in Huntingtin redistribution to the cell periphery, indicating dynein's role in transporting Huntingtin-associated vesicles toward the cell center . These established experimental paradigms provide a framework for new investigations using HTT4 antibody.

What are the optimal concentrations and conditions for using HTT4 antibody in different applications?

For optimal results with HTT4 (MW4-S) antibody, researchers should consider application-specific parameters. Based on validated protocols and manufacturer specifications, MW4-S is supplied at a concentration of approximately 0.02 μg/μl . For Western blot applications, researchers typically begin with dilutions in the range of 1:500-1:2000, though optimal dilutions should be determined empirically for each experimental system. For immunofluorescence applications, starting dilutions of 1:100-1:500 are generally recommended.

Critical experimental parameters to optimize include:

• Primary antibody incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

• Blocking reagents (BSA versus milk-based blockers)

• Washing stringency (buffer composition and number of washes)

• Detection methods (HRP-conjugated secondary antibodies versus fluorescent secondary antibodies)

Given the MW4-S concentration of 0.02 μg/μl, researchers should ensure they calculate dilutions accurately to maintain consistent antibody concentrations across experiments . For specialized applications, pilot experiments comparing multiple conditions are recommended to establish optimal parameters.

How does HTT4 antibody compare to recombinant monoclonal anti-Huntingtin antibodies in reproducibility and specificity?

When comparing MW4-S (a traditional mouse monoclonal) to newer recombinant monoclonal anti-Huntingtin antibodies, several important distinctions emerge. The comprehensive antibody characterization study evaluated traditional monoclonal antibodies alongside recombinant monoclonals like EPR5526, EP867Y, and D7F7 . Recombinant monoclonal antibodies often demonstrate improved lot-to-lot consistency due to their production method, which eliminates hybridoma drift issues that can affect traditional monoclonals like MW4-S.

The table below summarizes key characteristics of selected Huntingtin antibodies that researchers should consider when choosing between MW4-S and recombinant alternatives:

When selecting between these antibody types, researchers should consider their specific experimental requirements, including the need for lot-to-lot consistency, epitope recognition, and application-specific performance .

What troubleshooting approaches are recommended when HTT4 antibody shows poor signal-to-noise ratio?

When encountering poor signal-to-noise ratio with HTT4 antibody, researchers should implement a systematic troubleshooting approach:

• Validate antibody integrity: Confirm proper storage conditions and check for precipitation or contamination in the antibody solution. MW4-S has a concentration of approximately 0.02 μg/μl, which is relatively low compared to other antibodies, making proper handling critical .

• Optimize protein loading: For Western blot applications, adjust protein concentration based on the expression level of Huntingtin in the cell line. DMS 53 cells with higher HTT expression (6.1 log2 TPM+1) may require less protein loading compared to cell lines with lower expression like HAP1 (3.7 log2 TPM+1) .

• Adjust blocking conditions: Test alternative blocking agents (5% milk vs. BSA) and vary blocking times to reduce non-specific binding.

• Optimize antibody concentration: Perform a dilution series to identify the optimal antibody concentration that maximizes specific signal while minimizing background.

• Increase washing stringency: Implement additional washing steps or adjust buffer composition by increasing detergent concentration to reduce non-specific binding.

• Consider alternative detection methods: If using chemiluminescence, try fluorescent secondary antibodies which may provide better signal-to-noise ratios for quantitative analyses.

• Validate with positive and negative controls: Always include appropriate controls, ideally using knockout cell lines as negative controls to confirm signal specificity .

Implementation of these troubleshooting approaches should resolve most issues with poor signal-to-noise ratio when using HTT4 antibody.

How can HTT4 antibody be effectively used in multicolor immunofluorescence experiments?

For multicolor immunofluorescence experiments utilizing HTT4 (MW4-S) antibody, researchers should implement a strategic experimental design that accounts for species compatibility and spectral considerations:

• Antibody compatibility planning: Since MW4-S is a mouse monoclonal antibody, pair it with primary antibodies raised in different species (rabbit, goat, chicken) to allow for species-specific secondary antibody detection .

• Epitope availability assessment: When co-staining for Huntingtin and interacting partners like dynein, consider whether antibody binding might be mutually exclusive due to overlapping epitopes or conformational changes that occur during protein-protein interactions .

• Sequential staining protocol: For challenging combinations, implement sequential staining protocols using complete primary-secondary staining for one antibody followed by the second set.

• Spectral considerations: Choose fluorophores with minimal spectral overlap and optimize acquisition settings to prevent bleed-through between channels.

• Validation controls: Include single-antibody controls alongside multiplexed samples to confirm antibody specificity in the multiplexed context.

For studying Huntingtin's role in vesicular transport, researchers can design multicolor experiments combining MW4-S with antibodies against dynein/dynactin components and vesicular markers to visualize their spatial relationships during trafficking events . This approach would enable visualization of Huntingtin's colocalization with dynein and vesicular cargo in various cellular contexts.

What considerations should researchers take when using HTT4 antibody for quantitative Western blot analysis?

For quantitative Western blot analysis using HTT4 (MW4-S) antibody, researchers must implement rigorous methodological approaches to ensure accurate quantification:

When comparing wild-type and mutant Huntingtin with polyglutamine expansions, researchers should verify that MW4-S recognizes both forms with equal affinity to prevent quantification bias . Additionally, researchers should be aware that the large size of Huntingtin (~350 kDa) may require specialized transfer conditions and gel systems for optimal Western blot results.

How should researchers interpret conflicting results between different anti-HTT antibodies including HTT4?

When faced with conflicting results between different anti-HTT antibodies including HTT4 (MW4-S), researchers should implement a systematic approach to resolve discrepancies:

• Epitope mapping analysis: Compare the epitopes recognized by each antibody to determine if differences may be due to epitope accessibility in different experimental conditions or protein conformations. The MW series antibodies (including MW4-S) recognize different epitopes of Huntingtin .

• Antibody validation thoroughness: Evaluate the validation data available for each antibody, particularly focusing on knockout validation experiments. The comprehensive characterization study compared twenty Huntingtin antibodies using standardized protocols with knockout controls .

• Application-specific performance: Consider that antibodies may perform differently across applications (Western blot vs. immunofluorescence vs. immunoprecipitation). MW4-S is recommended for Western blot and immunofluorescence but not specifically for immunoprecipitation .

• Protein context considerations: Assess whether protein modifications, interactions, or conformational changes might differentially affect epitope accessibility across experimental conditions.

• Methodological differences: Examine differences in experimental protocols that might explain discrepancies, including fixation methods, detergent types, or buffer compositions.

To resolve conflicting results, researchers should implement parallel experiments using multiple antibodies together with appropriate negative controls (ideally knockout samples) . Additionally, employing orthogonal techniques that don't rely on antibodies (such as mass spectrometry or CRISPR tagging) can provide independent verification of results.

What are best practices for publishing research utilizing HTT4 antibody data?

When publishing research utilizing HTT4 (MW4-S) antibody data, researchers should follow these best practices to ensure reproducibility and transparency:

• Complete antibody reporting: Include comprehensive antibody details in methods sections, specifying catalog number (MW4-S), RRID (AB_528293), lot number, host species (mouse), and clonality (monoclonal) .

• Validation documentation: Describe antibody validation experiments performed, ideally including knockout validation results or reference previous validation studies like the comprehensive characterization study that tested twenty Huntingtin antibodies .

• Detailed methods documentation: Provide complete protocols including antibody dilutions, incubation times and temperatures, buffer compositions, and detection methods.

• Image acquisition parameters: For immunofluorescence data, document exposure settings, microscope specifications, and image processing methods.

• Quantification methods: Clearly describe quantification approaches, including software used, normalization methods, and statistical analyses performed.

• Replicate information: Specify the number of technical and biological replicates performed and how they were analyzed.

• Raw data availability: Consider making raw, unprocessed data available through appropriate repositories to enhance transparency and reproducibility.

By following these publication best practices, researchers can enhance the reproducibility of findings utilizing HTT4 antibody and facilitate comparison across studies in the Huntingtin research field.

How might HTT4 antibody be utilized in advanced imaging techniques for Huntingtin research?

HTT4 (MW4-S) antibody can be adapted for cutting-edge imaging approaches to advance Huntington's disease research through several innovative applications:

• Super-resolution microscopy: MW4-S can be utilized in techniques like STORM or STED microscopy to visualize Huntingtin localization with nanometer precision, potentially revealing previously undetectable subcellular distributions relevant to its function in vesicular transport .

• Live-cell imaging adaptations: Though direct MW4-S use in live cells is limited by cell permeability issues, researchers can develop derivative applications such as:

  • Developing MW4-S-derived single-chain variable fragments (scFvs) fused to fluorescent proteins for expression as intrabodies

  • Creating MW4-S-based SNAP-tag or HaloTag ligands for pulse-chase imaging of Huntingtin dynamics

• Proximity labeling applications: Combining MW4-S with enzyme-mediated proximity labeling techniques (BioID or APEX) to identify proteins in close proximity to Huntingtin in various cellular compartments.

• Correlative light and electron microscopy (CLEM): Using MW4-S for immunogold labeling to correlate fluorescence signals with ultrastructural features at the electron microscopy level, particularly relevant for studying Huntingtin's role in vesicular transport .

• Expansion microscopy compatibility: Validating MW4-S performance in expansion microscopy protocols to achieve super-resolution imaging on conventional microscopes through physical expansion of specimens.

These advanced imaging applications could particularly benefit studies of Huntingtin's role in dynein/dynactin-mediated vesicular transport, potentially revealing new insights into the spatial organization of these molecular complexes .

What recent advances in antibody engineering might improve future versions of HTT4 or similar antibodies?

Recent advances in antibody engineering offer several promising approaches that could enhance future versions of HTT4 or similar Huntingtin antibodies:

• Structure-guided antibody optimization: The emergence of accurate antibody loop structure prediction technologies enables zero-shot antibody design with higher specificity and affinity. Recent studies have demonstrated success rates of 13.2% for zero-shot antibody loop design, compared to previously reported rates of 1.8% . These approaches could be applied to optimize MW4-S variable regions for improved performance.

• Single-domain antibody developments: Converting conventional antibodies like MW4-S into single-domain formats (nanobodies or VHH antibodies) could improve tissue penetration for in vivo imaging and potentially cross the blood-brain barrier, which is particularly relevant for Huntington's disease research.

• Bispecific antibody formats: Engineering bispecific antibodies that simultaneously recognize Huntingtin and interaction partners like dynein could provide powerful tools for studying protein complexes involved in vesicular transport .

• Site-specific conjugation strategies: Developing site-specifically labeled MW4-S derivatives with precise fluorophore positioning to optimize FRET-based applications for detecting conformational changes in Huntingtin.

• Recombinant antibody production advancements: Transitioning from hybridoma-produced MW4-S to recombinant production systems would enhance consistency and enable genetic engineering for improved performance characteristics. The comprehensive antibody characterization study included several recombinant monoclonal antibodies that could serve as models for this approach .

These engineering advances could significantly enhance the utility of Huntingtin antibodies for both basic research and potential therapeutic applications in Huntington's disease.