HLTF Antibody, HRP conjugated

What is HLTF and what are its primary functions in cellular biology?

HLTF is a multifunctional protein that possesses both helicase and E3 ubiquitin ligase activities. It has intrinsic ATP-dependent nucleosome-remodeling activity that may be required for transcriptional activation or repression of specific target promoters. HLTF plays a critical role in error-free postreplication repair (PRR) of damaged DNA and maintains genomic stability through acting as a ubiquitin ligase for 'Lys-63'-linked polyubiquitination of chromatin-bound PCNA (Proliferating Cell Nuclear Antigen) .

HLTF shares structural features with yeast Rad5, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. The protein can interact with DNA and functions in a pathway that promotes error-free replication through DNA lesions .

Why are HRP-conjugated antibodies commonly used in research applications?

HRP-conjugated antibodies provide several methodological advantages in research applications:

• Signal amplification: HRP catalyzes reactions that produce detectable signals (colorimetric, chemiluminescent, or fluorescent), significantly enhancing detection sensitivity.

• Stability: HRP-conjugated antibodies demonstrate good stability when properly stored, maintaining enzymatic activity for extended periods.

• Versatility: These conjugates can be used across multiple applications including ELISA, Western blotting, immunohistochemistry, and other immunoassays .

• Compatibility: HRP works well with numerous substrates, allowing researchers to select the optimal detection method for their experimental requirements .

What applications are most suitable for HLTF antibody, HRP-conjugated?

HLTF antibody conjugated to HRP is particularly suitable for:

• Western Blotting (WB): For detecting HLTF protein expression in cell or tissue lysates

• ELISA: For quantitative determination of HLTF levels

• Immunocytochemistry (ICC): For cellular localization studies of HLTF

• Immunohistochemistry (IHC): For tissue-based detection of HLTF

For optimal results, researchers should validate the specific antibody for their application of interest and sample type (human, mouse, etc.) as reactivity may vary between products .

What are the advantages of using an HRP-conjugated HLTF antibody versus a two-step detection system?

Methodological comparison:

For HLTF detection specifically, direct HRP conjugation is advantageous when:

• Reducing experimental time is critical

• Background is problematic in your system

• Cross-reactivity with secondary antibodies has been observed

• The target is sufficiently abundant to be detected without signal amplification

How can I optimize HRP-conjugated antibody signal in Western blotting when detecting HLTF?

Optimizing HRP-conjugated HLTF antibody signals requires attention to several methodological factors:

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers) to determine which provides the best signal-to-noise ratio for HLTF detection.

• Antibody concentration: Titrate the HRP-conjugated HLTF antibody to find the optimal dilution that maximizes specific signal while minimizing background. Starting ranges typically between 1:1000-1:5000 should be tested.

• Incubation conditions:

  • Temperature: Compare room temperature vs. 4°C incubation

  • Time: Test different durations (1 hour to overnight)

  • Buffer composition: Optimize detergent concentration (0.05-0.1% Tween-20)

• Wash stringency: Adjust wash buffer composition and duration to remove non-specific binding while preserving specific signals.

• Substrate selection: HRP substrates vary in sensitivity:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Advanced ECL substrates for higher sensitivity

  • Colorimetric substrates for qualitative analysis

• Exposure optimization: For chemiluminescent detection, capture multiple exposure times to identify the optimal signal window .

What are the best methods for preparing custom HRP-conjugated HLTF antibodies?

Several methodological approaches can be employed for preparing custom HRP-conjugated HLTF antibodies:

• Enhanced Periodate Method with Lyophilization:

  • Activate HRP using 0.15M sodium metaperiodate

  • Dialyze against PBS

  • Freeze at -80°C and lyophilize overnight

  • Mix with HLTF antibody (1:4 molar ratio of antibody:HRP)

  • Incubate at 37°C for 1 hour

  • Stabilize with sodium cyanoborohydride (1/10th volume)

  • Dialyze against PBS

  This modified method has demonstrated significantly improved sensitivity (1:5000 dilution) compared to classical methods (1:25 dilution) .

• LYNX Rapid Conjugation Kit Approach:

  • Using commercial kits that contain pre-prepared lyophilized HRP

  • Adding modifier reagent to antibody solution

  • Combining with lyophilized HRP

  • Adding quencher after incubation

  This method offers 100% antibody recovery and maintains near-neutral pH during conjugation .

• Glutaraldehyde Method:

  • Two-step process using glutaraldehyde as a crosslinker

  • Requires careful pH control and ratio optimization

  • Offers good stability but potentially lower activity than periodate method

Confirmation of successful conjugation should be performed using:

• UV spectrophotometry (characteristic peaks at 280nm for antibody and 430nm for HRP)

• SDS-PAGE analysis (conjugates show mobility shifts) .

How does HLTF expression correlate with cancer progression, and how can HRP-conjugated antibodies help investigate this relationship?

HLTF exhibits context-dependent roles in cancer biology:

• Tumor suppressor function:

  • HLTF is frequently inactivated in colorectal and gastric cancers through promoter hypermethylation

  • Low HLTF expression is associated with poor prognosis in lung cancer and certain melanomas

  • In hepatocellular carcinoma (HCC), decreased HLTF expression results in aggressive clinicopathological features

• Paradoxical oncogenic role:

  • Upregulation of HLTF has been reported in hypopharyngeal and cervical cancers

  • HLTF activation is linked to estrogen-induced renal carcinogenesis

HRP-conjugated HLTF antibodies provide valuable methodological approaches for investigating these relationships:

• Quantitative tissue analysis: Immunohistochemistry using HRP-conjugated HLTF antibodies allows visualization of expression patterns across different tumor stages and grades.

• Protein-protein interaction studies: HRP-conjugated HLTF antibodies can be used in co-immunoprecipitation experiments to identify interaction partners in cancer cells.

• Chromatin binding studies: ChIP assays using HRP-conjugated HLTF antibodies can map genomic binding sites in normal versus cancer cells.

• Signaling pathway analysis: Western blotting with HRP-conjugated HLTF antibodies can track expression changes in response to therapeutic interventions or genetic manipulations .

What is the role of HLTF in the β-TrCP/HLTF/p62/mTOR signaling axis and how can this be studied using HRP-conjugated antibodies?

Research has identified a critical β-TrCP/HLTF/p62/mTOR signaling axis, particularly in hepatocellular carcinoma:

• β-TrCP (FBXW1) is an E3 ubiquitin ligase that recognizes HLTF through a specific binding motif ('DSXXXS')

• β-TrCP mediates polyubiquitination and subsequent degradation of HLTF

• Decreased HLTF levels facilitate p62 transcription and activate the mTOR pathway

• This pathway promotes HCC cell proliferation and metastasis

Methodological approaches using HRP-conjugated antibodies to study this pathway include:

• In vivo ubiquitination assays: Using HRP-conjugated antibodies to detect ubiquitinated forms of HLTF after immunoprecipitation.

• Protein stability assessments: Pulse-chase experiments with cycloheximide treatment followed by Western blotting with HRP-conjugated HLTF antibodies to determine protein half-life.

• Phosphorylation analysis: Investigating S544/S548 phosphorylation status (the β-TrCP binding sites) using phospho-specific antibodies alongside HRP-conjugated total HLTF antibodies.

• Mutation impact studies: Comparing wild-type versus S544R/S548R mutant HLTF expression using HRP-conjugated antibodies that recognize both forms.

• Downstream signaling analysis: Multiplex Western blotting using HRP-conjugated antibodies against HLTF, p62, and phosphorylated mTOR pathway components .

How can I detect HLTF in chromatin fractions versus soluble nuclear fractions using HRP-conjugated antibodies?

Differential detection of HLTF in chromatin versus soluble nuclear fractions requires careful subcellular fractionation followed by sensitive detection:

Methodological procedure:

• Cell fractionation protocol:

  • Isolate nuclei using hypotonic lysis buffer

  • Extract soluble nuclear fraction using low-salt buffer

  • Isolate chromatin-bound proteins using high-salt extraction or nuclease treatment

  • Confirm fraction purity using markers (histone H3 for chromatin, PARP for soluble nuclear)

• Western blotting optimization:

  • Load equivalent protein amounts from each fraction

  • Include appropriate fraction-specific controls

  • Use HRP-conjugated HLTF antibody at optimal dilution (typically 1:1000-1:2000)

  • For chromatin fractions, ensure complete solubilization using appropriate buffers

• Signal enhancement strategies:

  • Use advanced ECL substrates for low-abundance detection

  • Consider tyramide signal amplification for immunohistochemistry applications

  • Optimize exposure times separately for each fraction

• Quantification approach:

  • Normalize HLTF signals to fraction-specific loading controls

  • Calculate chromatin/soluble ratios to assess distribution

  • Compare across experimental conditions to detect shifts in localization

This approach is particularly valuable for studying HLTF's dual roles in transcriptional regulation and DNA repair, as its distribution between chromatin and soluble fractions may change in response to DNA damage or other cellular stresses.

What are common causes of high background when using HRP-conjugated HLTF antibodies and how can they be addressed?

High background is a frequent challenge when using HRP-conjugated antibodies. For HLTF detection specifically, consider these methodological solutions:

How can I distinguish between specific and non-specific signals when detecting HLTF with HRP-conjugated antibodies?

Distinguishing specific from non-specific signals requires rigorous controls and validation approaches:

• Essential controls:

  • Knockout/knockdown validation: Compare signals between HLTF knockout/knockdown samples and wild-type samples

  • Peptide competition: Pre-incubate antibody with excess immunizing peptide to block specific binding

  • Secondary-only control: For indirect detection methods

  • Isotype control: Use an irrelevant HRP-conjugated antibody of the same isotype

• Technical validation approaches:

  • Multiple antibodies: Confirm results using antibodies targeting different HLTF epitopes

  • Molecular weight verification: Ensure signal appears at the expected molecular weight (~114 kDa for full-length HLTF)

  • Signal pattern analysis: Compare to known HLTF expression patterns in tissues/cells

  • Reciprocal verification: Confirm results using an alternative detection method (e.g., fluorescence)

• Advanced validation methods:

  • Immunoprecipitation-Western blot: Perform IP with one antibody and detect with another

  • Mass spectrometry validation: Confirm protein identity by mass spectrometry after immunoprecipitation

  • Correlation with mRNA levels: Compare protein detection with RT-qPCR results

How do different fixation methods affect HLTF epitope accessibility when using HRP-conjugated antibodies for immunohistochemistry?

Fixation significantly impacts epitope accessibility and preservation when detecting HLTF in tissues:

Methodological comparison of fixation methods:

For optimal HLTF detection specifically:

• Use 10% NBF for no more than 24 hours

• Process tissues promptly to embedded blocks

• Perform HIER with EDTA buffer (pH 9.0)

• Consider enzyme-based retrieval as an alternative approach

• Optimize antibody dilution for each fixation method

• Include properly fixed positive control tissues

What unique challenges exist when detecting phosphorylated forms of HLTF using HRP-conjugated antibodies?

Detecting phosphorylated HLTF forms (particularly at S544/S548 sites implicated in β-TrCP recognition) presents specific methodological challenges:

• Rapid dephosphorylation:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers

  • Process samples rapidly at cold temperatures

  • Consider protein extraction directly into hot SDS buffer for immediate denaturation

• Low abundance issues:

  • Enrich phosphorylated forms using phospho-specific antibodies for immunoprecipitation

  • Use phospho-enrichment techniques (IMAC, TiO₂) prior to detection

  • Consider signal amplification systems for detection

• Specificity validation:

  • Treatment with lambda phosphatase as negative control

  • Use of phosphomimetic (S→D/E) and non-phosphorylatable (S→A) mutants as controls

  • Validation with mass spectrometry

• Detection optimization:

  • Optimize membranes (PVDF typically retains phosphoproteins better than nitrocellulose)

  • Use milk-free blocking reagents (milk contains phosphatases)

  • Consider using phospho-specific HLTF antibodies followed by HRP-conjugated secondary antibodies for higher specificity

• Signaling context:

  • Monitor S6K activity (the kinase responsible for S544/S548 phosphorylation)

  • Use S6K inhibitors (e.g., PF-4708671) as controls

  • Compare wild-type versus S544R/S548R mutant HLTF phosphorylation patterns

How has HRP-conjugated HLTF antibody been used to study the role of HLTF in DNA damage response pathways?

HRP-conjugated HLTF antibodies have been instrumental in elucidating HLTF's role in DNA damage response pathways through several methodological applications:

• PCNA polyubiquitination studies:

  • HRP-conjugated HLTF antibodies were used to detect HLTF recruitment to chromatin following DNA damage

  • HLTF was shown to physically interact with Rad6-Rad18 and Mms2-Ubc13 complexes

  • These studies demonstrated HLTF's role in promoting Lys-63-linked polyubiquitination of PCNA at Lys-164

• Error-free postreplication repair pathway analysis:

  • Western blotting with HRP-conjugated HLTF antibodies helped establish HLTF as a functional homolog of yeast Rad5

  • These studies showed that HLTF inactivation renders human cells sensitive to UV and other DNA-damaging agents

  • The research confirmed that HLTF can complement the UV sensitivity of rad5Δ yeast strains

• Protein complex identification:

  • Co-immunoprecipitation followed by detection with HRP-conjugated antibodies revealed that HLTF associates with PCNA, Ubc13, Mms2, and Rad18

  • These interactions were shown to occur even in the absence of UV irradiation, suggesting constitutive complex formation

How can HRP-conjugated antibodies be used to study the relationship between HLTF and T follicular helper (Tfh) cells in immune responses?

While HLTF and Tfh cells represent distinct biological entities, methodological approaches using HRP-conjugated antibodies can help investigate their potential relationships:

• Transcription factor network analysis:

  • HLTF as a transcription factor may regulate genes involved in Tfh differentiation

  • ChIP-sequencing using HRP-conjugated HLTF antibodies can identify HLTF binding sites in Tfh-related gene promoters

  • Western blotting can examine HLTF expression during Tfh cell differentiation stages

• HLTF expression in immune cell subsets:

  • Flow cytometry with HRP-conjugated antibodies (using tyramide signal amplification) can assess HLTF expression in different T cell populations including Tfh cells

  • Immunohistochemistry can visualize HLTF expression in germinal centers where Tfh cells reside

• Functional relationships in disease models:

  • In COVID-19 research, where Tfh cells have been observed to be lost in severe cases while antibody production continues

  • HRP-conjugated HLTF antibodies could help investigate alternative transcriptional networks operating in non-canonical antibody production pathways

  • This could elucidate whether HLTF plays a role in Tfh-independent antibody responses observed in certain viral infections

What are the most sensitive detection methods when working with HRP-conjugated HLTF antibodies for low-abundance targets?

When detecting low-abundance HLTF or studying contexts where HLTF expression is reduced, several methodological approaches can enhance sensitivity:

• Enhanced chemiluminescence (ECL) optimization:

  • Super Signal West Femto substrate provides femtogram-level detection

  • Incubation time optimization (typically 3-5 minutes)

  • Multiple short exposures to prevent signal saturation

  • Dark-adapted CCD cameras for digital acquisition

• Tyramide signal amplification (TSA):

  • Enhances sensitivity 10-100 fold over conventional HRP detection

  • HRP catalyzes deposition of fluorescent or chromogenic tyramide

  • Particularly valuable for tissue immunohistochemistry with low HLTF expression

  • Can be multiplexed with other markers for co-localization studies

• Capillary Western technologies:

  • Systems like Jess or Wes (ProteinSimple) offer higher sensitivity than traditional Western blotting

  • Require smaller sample volumes (as little as 3μL)

  • Quantitative results with broader dynamic range

  • Automated process reduces variability

• Sample preparation enhancements:

  • Nuclear extraction to concentrate HLTF

  • Immunoprecipitation before Western blotting

  • Proteasome inhibitors (MG132) to prevent degradation of ubiquitinated HLTF

  • Phosphatase inhibitors to preserve post-translational modifications

• Microfluidic immunoassays:

  • Confined reaction spaces increase effective concentration

  • Reduced diffusion distances improve kinetics

  • Lower sample consumption with higher sensitivity

How might advances in HRP conjugation technology improve detection of HLTF protein isoforms or post-translational modifications?

Emerging advances in HRP conjugation technology present exciting opportunities for studying HLTF biology:

• Site-specific conjugation methods:

  • Enzymatic conjugation using sortase A or formylglycine-generating enzyme

  • Click chemistry approaches using copper-free click reactions

  • These methods create more homogeneous conjugates with preserved antigen binding

• Enhanced HRP variants:

  • Engineered HRP enzymes with improved stability and catalytic efficiency

  • Temperature-resistant variants for more stringent wash conditions

  • Reduced glycosylation variants to minimize non-specific binding

• Applications for HLTF research:

  • Improved detection of specific HLTF isoforms through epitope-preserving conjugation

  • Higher sensitivity for detecting transient post-translational modifications (phosphorylation at S544/S548)

  • More precise quantification of HLTF ubiquitination status

  • Better discrimination between HLTF conformational states

• Multiplex detection systems:

  • Spectrally distinct HRP substrates for simultaneous detection of HLTF and interaction partners

  • Quantum dot-mediated HRP reactions for improved signal-to-noise ratios

  • Integration with mass cytometry for single-cell analysis of HLTF status

What methodological approaches could be developed to study HLTF's dual roles in transcription regulation and DNA repair simultaneously?

Investigating HLTF's dual functionality requires sophisticated methodological approaches:

• Chromatin dynamics analysis:

  • Proximity ligation assays with HRP-conjugated antibodies to visualize HLTF interactions with transcription versus repair machinery

  • ChIP-sequencing during normal conditions versus DNA damage to map changing genomic binding sites

  • Live-cell imaging using split-HRP systems to monitor dynamic HLTF relocalization

• Domain-specific functional studies:

  • Generate domain-specific antibodies (HIRAN domain, RING domain, helicase domain) with HRP conjugation

  • Map domain-specific interactions during transcription versus repair functions

  • Correlate with functional outcomes using reporter systems

• Temporal analysis systems:

  • Synchronize cells and use HRP-conjugated HLTF antibodies to track protein dynamics through cell cycle

  • Pulse-chase experiments to determine if distinct HLTF pools participate in different functions

  • Single-molecule tracking with HRP-based detection to follow individual HLTF molecules

• Proteomics integration:

  • Develop HRP-APEX fusion systems for proximity labeling of HLTF interactors

  • Compare interactome during transcriptional activation versus DNA damage response

  • Identify post-translational modifications that direct HLTF toward specific functions

What potential exists for developing therapeutic antibodies targeting HLTF, and how might HRP conjugation facilitate this research?

The potential for HLTF-targeted therapeutics represents an emerging research direction:

• Target validation approaches:

  • HRP-conjugated antibodies can quantify HLTF expression across cancer types

  • Immunohistochemistry with HRP detection to correlate HLTF levels with prognosis

  • Patient stratification based on HLTF status to identify suitable candidates for targeted therapy

• Functional antibody screening:

  • HRP-based assays to identify antibodies that modulate HLTF's E3 ubiquitin ligase activity

  • ELISA systems using HRP-conjugated antibodies to screen for compounds that stabilize HLTF

  • High-throughput imaging with HRP-based detection to assess effects on HLTF localization

• Antibody-drug conjugate (ADC) development:

  • For cancers where HLTF is overexpressed, HRP conjugation methods can inform optimal conjugation sites

  • Stability testing of various linkage chemistries using HRP-based detection methods

  • Internalization assays with HRP substrates to track ADC trafficking

• Combination therapy research:

  • HRP-conjugated antibodies to assess HLTF modulation during conventional therapies

  • Identification of synergistic drug combinations that restore HLTF function in tumors

  • Biomarker development to monitor therapeutic efficacy in real-time