HINFP Antibody, HRP conjugated

What is HINFP and what is its biological function?

HINFP (Histone H4 Transcription Factor) is a multi-functional transcription regulator with several important biological roles. It functions as a transcriptional repressor that binds to the consensus sequence 5'-CGGACGTT-3' and to the RB1 promoter. It also acts as a transcriptional activator that promotes histone H4 gene transcription at the G1/S phase transition in conjunction with NPAT. Additionally, HINFP activates transcription of the ATM and PRKDC genes and autoregulates its expression by associating with its own promoter .

Research has also demonstrated that HINFP (also known as MIZF) interacts with MBD2, a methyl-CpG-binding protein that plays a critical role in DNA methylation-mediated transcriptional repression. In this capacity, HINFP functions as a negative regulator of transcription by binding to MBD2 and recruiting histone deacetylase complexes .

What is an HRP-conjugated antibody and how does it work in immunoassays?

An HRP-conjugated antibody is an immunoglobulin molecule that has been chemically linked to horseradish peroxidase (HRP), an enzyme that catalyzes the oxidation of substrates by hydrogen peroxide, producing a detectable signal. In the case of HINFP Antibody-HRP conjugates, the antibody portion binds specifically to HINFP protein while the HRP portion provides a means of detection.

When used in immunoassays such as ELISA, the antibody-HRP conjugate binds to the target antigen (HINFP), and upon addition of an appropriate substrate, the HRP enzyme catalyzes a reaction that produces a colorimetric, chemiluminescent, or fluorescent signal. This signal can be measured to quantify the amount of HINFP present in the sample . The sensitivity of detection depends on various factors, including the quality of conjugation between the antibody and HRP, which can be optimized through methods such as lyophilization during the conjugation process .

What are the standard applications for HINFP Antibody, HRP conjugated?

HINFP Antibody, HRP conjugated is primarily used in the following research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary application where HRP-conjugated HINFP antibodies are used for direct detection of HINFP in samples with recommended dilutions typically ranging from 1:500 to 1:1000 .

• Western Blot: While unconjugated primary antibodies are more commonly used for Western blotting, HRP-conjugated antibodies can sometimes be used in direct detection systems.

• Immunocytochemistry/Immunofluorescence: For visualizing the cellular localization of HINFP, particularly in human cell lines such as HeLa and L02 cells .

• Immunohistochemistry: For detecting HINFP in tissue sections, including human, mouse, and rat liver tissues .

What dilution factors should be used when working with HINFP Antibody, HRP conjugated?

Optimal dilution factors for HINFP Antibody, HRP conjugated vary based on:

For ELISA applications:

• Standard recommended dilution: 1:500-1:1000

• Optimized protocols with enhanced conjugation methods may allow for dilutions up to 1:5000, significantly more sensitive than classical conjugation methods which may only work at dilutions as low as 1:25

Important considerations:

• Dilution factors should be determined empirically for each specific application and experimental condition

• Titration experiments are recommended when using the antibody in a new protocol or with different sample types

• Enhanced conjugation methods using lyophilization may allow for more economical use of the antibody through higher dilution factors

How should HINFP Antibody, HRP conjugated be stored to maintain optimal activity?

Proper storage is crucial for preserving the activity of both the antibody and the HRP enzyme:

• Temperature: Aliquot and store at -20°C for long-term storage

• Light exposure: Avoid exposure to light as HRP is light-sensitive

• Freeze/thaw cycles: Avoid repeated freeze/thaw cycles which can degrade both the antibody and enzyme activity

• Buffer composition: Typically stored in 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol

• Aliquoting: Divide into small aliquots before freezing to avoid repeated freeze/thaw cycles; 20 μl sizes may contain 0.1% BSA for additional stability

When properly stored, the conjugated antibody should maintain its activity for at least one year after shipment .

What controls should be included when using HINFP Antibody, HRP conjugated in experiments?

Rigorous experimental design requires appropriate controls when using HINFP Antibody, HRP conjugated:

Positive controls:

• Known HINFP-expressing samples: HepG2 cells, human liver tissue, mouse liver tissue, rat liver tissue, and L02 cells have been validated to express detectable levels of HINFP

Negative controls:

• Isotype control: Rabbit IgG-HRP conjugate with no specific target to assess non-specific binding

• Antigen blocking: Pre-incubation of the antibody with recombinant HINFP protein (specifically the 370-515AA region used as immunogen)

• HRP substrate only: To check for endogenous peroxidase activity or non-specific substrate reactions

Specificity controls:

• Knockdown/knockout validation: HINFP knockdown or knockout samples to confirm signal specificity

• Cross-reactivity assessment: Testing on samples from different species to confirm the human-specific reactivity

How can lyophilization enhance the conjugation efficiency of HRP to HINFP antibodies?

The introduction of a lyophilization step in the HRP-antibody conjugation process represents a significant methodological advancement that researchers should consider:

Lyophilization during conjugation offers several advantages:

• Enhanced binding capacity: The process allows antibodies to bind more HRP molecules, creating conjugates with higher enzyme-to-antibody ratios

• Mechanism of enhancement:

  • Sodium meta periodate is used to oxidize carbohydrate moieties on HRP, generating aldehyde groups

  • The activated form of HRP is then lyophilized, creating a stable intermediate

  • When mixed with antibodies (typically at 1 mg/ml concentration), the lyophilized HRP shows enhanced conjugation efficiency

• Performance improvement: Conjugates prepared using lyophilization can perform efficiently at dilutions as high as 1:5000, whereas traditional conjugation methods may only work effectively at much lower dilutions around 1:25

• Practical significance: This technical modification enables:

  • Reduced antibody consumption in experiments

  • Improved sensitivity in immunoassays

  • More cost-effective research practices

  • Potential for detecting lower abundance targets

Researchers working with HINFP should consider either purchasing commercially available enhanced conjugates or implementing this lyophilization step when preparing their own HRP-antibody conjugates .

What are the molecular weights and protein characteristics to consider when analyzing HINFP using antibody detection methods?

Understanding the molecular properties of HINFP is essential for accurate experimental design and data interpretation:

Molecular weight considerations:

• Calculated molecular weight: 60 kDa

• Observed molecular weight: 60-70 kDa in SDS-PAGE analysis

• Migration patterns may vary depending on post-translational modifications

• Possible detection of multiple bands representing different isoforms

HINFP protein characteristics:

• UniProt Primary Accession: Q9BQA5

• UniProt Secondary Accessions: B3KPH6, B4DWB4, E9PQF4, Q96E65, Q9Y4M7

• UniProt Entry Name: HINFP_HUMAN

• Gene Symbol: HINFP (also known as MIZF, ZNF743)

• GeneID: 25988

• GenBank Accession Number: BC001073

• String identifier: 9606.ENSP00000318085

Alternative names and isoforms:

• Histone H4 transcription factor

• Histone nuclear factor P (HiNF-P)

• MBD2-interacting zinc finger protein (MIZF)

• Methyl-CpG-binding protein 2-interacting zinc finger protein

• ZNF743

This information is particularly important when analyzing Western blots, immunoprecipitation results, or mass spectrometry data involving HINFP detection.

How can multiplex detection approaches be optimized when working with HINFP Antibody, HRP conjugated?

Advanced research often requires simultaneous detection of multiple targets. When incorporating HINFP Antibody, HRP conjugated into multiplex approaches:

Cocktail antibody strategies:

• Similar to the approach used with oligonucleotide probes, using a cocktail of antibodies targeting different epitopes of HINFP can increase sensitivity

• Consider combining the HRP-conjugated HINFP antibody with antibodies targeting known HINFP-interacting proteins like NPAT, MBD2, or components of histone deacetylase complexes

Multiplex immunoassay optimization:

• Substrate selection: Choose HRP substrates compatible with other detection systems:

  • TMB (3,3',5,5'-tetramethylbenzidine) for colorimetric detection

  • Luminol-based reagents for chemiluminescence

  • Tyramide signal amplification for fluorescence multiplexing

• Cross-reactivity prevention:

  • Thoroughly validate antibody specificity before multiplex experiments

  • Use careful blocking and washing protocols to minimize background

  • Consider sequential detection approaches rather than simultaneous detection if cross-reactivity occurs

• Signal separation:

  • For fluorescence applications, ensure appropriate spectral separation between fluorophores

  • For chromogenic applications, use distinct substrates that produce different colored products

  • Consider compartmentalized detection (nuclear vs. cytoplasmic) based on HINFP's nuclear localization

What are common causes of false positives/negatives when using HINFP Antibody, HRP conjugated, and how can they be addressed?

Common causes of false positives:

Common causes of false negatives:

How can researchers distinguish between specific and non-specific binding when using HINFP Antibody, HRP conjugated?

Distinguishing specific from non-specific binding requires systematic validation approaches:

• Control experiments:

  • Peptide competition/blocking: Pre-incubate antibody with recombinant HINFP protein (370-515AA region) to block specific binding sites

  • Gradient dilution series: Specific signals typically diminish proportionally with dilution

  • Isotype controls: Use matched IgG-HRP conjugate at equivalent concentration

• Signal pattern analysis:

  • Specific binding should correspond to known molecular weight (60-70 kDa)

  • Specific signals should show expected subcellular localization (nuclear for HINFP)

  • Compare with literature or database patterns of HINFP expression

• Alternative detection methods:

  • Confirm findings using a second antibody targeting a different epitope of HINFP

  • Validate with orthogonal techniques (qPCR, mass spectrometry)

  • Employ genetic approaches (siRNA knockdown, CRISPR knockout)

• Quantitative assessment:

  • Calculate signal-to-noise ratios under different conditions

  • Use image analysis software to quantify signal intensity and distribution

  • Compare results with predicted expression patterns based on tissue/cell type

What strategies can address batch-to-batch variability in HINFP Antibody, HRP conjugated preparations?

Batch-to-batch variability is a significant challenge in antibody-based research. Strategies to address this include:

• Standardization protocols:

  • Establish internal validation benchmarks for each new antibody batch

  • Use the same positive control samples across experiments

  • Develop quantitative metrics for antibody performance (sensitivity, specificity)

• Calibration approaches:

  • Create standard curves with recombinant HINFP protein

  • Normalize signals to housekeeping proteins or internal controls

  • Use reference samples with known HINFP concentration

• Enhanced conjugation methods:

  • Consider using lyophilization-enhanced conjugation for more consistent results

  • Document conjugation ratios and enzymatic activity for each batch

  • Test multiple dilutions to determine optimal working concentration

• Long-term planning:

  • Purchase multiple vials from the same lot for long-term studies

  • Aliquot antibodies to avoid freeze-thaw issues

  • Consider developing stable cell lines expressing tagged HINFP as consistent controls

How might HINFP Antibody, HRP conjugated be applied in studying epigenetic regulation mechanisms?

Given HINFP's role in transcriptional regulation and interaction with DNA methylation machinery, several advanced applications emerge:

• Investigating HINFP in DNA methylation pathways:

  • ChIP-seq with HINFP antibodies to map genomic binding sites

  • Co-immunoprecipitation to isolate HINFP-MBD2 complexes

  • Analysis of HINFP recruitment to methylated promoters

• Cell cycle-dependent histone regulation:

  • Synchronized cell studies to examine HINFP's role in histone H4 transcription at G1/S transition

  • Correlation between HINFP binding and histone modification patterns

  • Investigation of HINFP's interaction with NPAT in cell cycle control

• Autoregulatory mechanisms:

  • Analysis of HINFP's binding to its own promoter

  • Investigation of feedback loops in transcriptional regulation

  • Study of post-translational modifications affecting HINFP activity

• Methodological approaches:

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq)

  • Proximity ligation assays to study HINFP-protein interactions in situ

  • CRISPR-mediated genomic editing to study HINFP binding site mutations

What are the implications of HINFP's role in transcriptional regulation for cancer research?

HINFP's roles in transcriptional regulation, cell cycle control, and epigenetic processes suggest important connections to cancer biology:

• HINFP in proliferation control:

  • Investigation of HINFP levels in rapidly dividing cancer cells

  • Analysis of histone H4 regulation in response to oncogenic signals

  • Study of HINFP's interaction with cell cycle regulators

• RB1 promoter regulation:

  • Given HINFP's binding to the RB1 promoter , investigation of retinoblastoma protein expression modulation

  • Analysis of HINFP mutations or expression changes in retinoblastoma and other cancers

  • Therapeutic targeting potential by modulating HINFP-mediated transcriptional repression

• DNA damage response pathway:

  • Study of HINFP's activation of ATM and PRKDC genes in DNA damage response

  • Investigation of HINFP as a potential biomarker for DNA damage sensitivity

  • Analysis of HINFP's role in chemotherapy resistance mechanisms

• Epigenetic dysregulation:

  • Examination of altered HINFP-MBD2 interactions in cancer cells

  • Study of aberrant DNA methylation patterns in relation to HINFP binding

  • Investigation of histone deacetylase recruitment by HINFP in tumor suppressor silencing

How can advanced imaging techniques be combined with HINFP Antibody, HRP conjugated for subcellular localization studies?

Combining advanced imaging with HRP-conjugated antibodies opens new research avenues:

• Super-resolution microscopy applications:

  • Tyramide signal amplification (TSA) with HRP-conjugated HINFP antibodies for high-sensitivity detection

  • Correlative light and electron microscopy (CLEM) to visualize HINFP at ultrastructural level

  • Multi-color imaging with spectral unmixing to study HINFP co-localization with interaction partners

• Live-cell imaging strategies:

  • HRP-SNAP tag combinations for pulse-chase experiments

  • Proximity labeling approaches using HRP to identify proteins in close proximity to HINFP

  • Development of cell-permeable substrates for intracellular HRP detection

• Quantitative image analysis:

  • Digital image processing for precise nuclear vs. cytoplasmic distribution

  • Machine learning approaches for pattern recognition in HINFP localization

  • Correlation of HINFP distribution with cell cycle phases or treatment responses

• Methodological considerations:

  • Optimization of fixation and permeabilization for epitope preservation

  • Balance between signal amplification and spatial resolution

  • Combined use of fluorescent and chromogenic detection for multiplexed imaging

How does the sensitivity of HRP-conjugated HINFP antibodies compare with other detection systems?

Understanding the relative advantages of different detection approaches is crucial for experimental design:

The enhanced HRP-conjugated antibodies prepared using lyophilization during conjugation can achieve sensitivities allowing dilutions up to 1:5000, significantly outperforming traditional conjugation methods that typically work at 1:25 dilutions .

What considerations should guide the choice between direct HRP-conjugated antibodies versus two-step detection methods for HINFP?

Selecting between direct (HRP-conjugated primary antibody) and indirect (primary antibody + HRP-conjugated secondary) detection requires balancing several factors:

Advantages of direct detection with HRP-conjugated HINFP antibodies:

• Simplified workflow with fewer incubation and washing steps

• Reduced background from non-specific secondary antibody binding

• Better for multiplex detection when antibodies from the same species are used

• Shorter total protocol time

• Enhanced conjugation methods (e.g., lyophilization) may provide sufficient sensitivity

Advantages of indirect (two-step) detection:

• Signal amplification through multiple secondary antibodies binding each primary

• Greater flexibility as the same secondary can be used with multiple primaries

• More economical for multiple targets (only need HRP-conjugated secondary)

• Often higher sensitivity for low-abundance targets

• Primary antibody concentration can be higher without increased cost

Decision factors:

• Target abundance (low abundance may benefit from indirect detection)

• Experimental constraints (time limitations favor direct detection)

• Multiplexing requirements (direct detection simplifies same-species antibody use)

• Budget considerations (indirect methods may be more economical for multiple targets)

• Background concerns (direct methods may reduce non-specific binding)

How might advances in antibody engineering and conjugation chemistry improve HINFP detection methods?

Emerging technologies suggest several promising directions for enhancing HINFP antibody-based detection:

• Site-specific conjugation:

  • Engineered antibodies with site-specific attachment points for HRP

  • Consistent enzyme-to-antibody ratios for better reproducibility

  • Preserved antigen-binding regions for optimal target recognition

• Alternative enzymes and reporters:

  • Engineered HRP variants with enhanced stability or catalytic efficiency

  • Alternative enzymes like alkaline phosphatase with distinct advantages

  • Nanobody-enzyme fusions for improved tissue penetration and reduced size

• Conjugation chemistry innovations:

  • Click chemistry approaches for efficient, specific conjugation

  • Hydrogel or dendrimeric scaffolds to increase enzyme loading

  • Building on lyophilization techniques with additional stabilizing agents

• Recombinant antibody formats:

  • Single-chain variable fragments (scFvs) against HINFP with direct HRP fusion

  • Bispecific antibodies combining HINFP targeting with binding to interaction partners

  • Intracellular antibodies (intrabodies) for live-cell applications

What are emerging applications combining HINFP detection with other molecular biology techniques?

Integration of HINFP antibody detection with cutting-edge molecular techniques offers new research opportunities:

• Spatial transcriptomics:

  • Combining HINFP protein detection with RNA-seq data in tissue sections

  • Correlation of HINFP localization with target gene expression patterns

  • Integration with single-cell approaches for heterogeneity analysis

• CRISPR screening applications:

  • High-throughput screening of factors affecting HINFP expression or localization

  • Monitoring HINFP levels as a readout for epigenetic regulators

  • Creating reporter systems based on HINFP transcriptional activity

• Protein interaction mapping:

  • Proximity labeling combined with mass spectrometry for HINFP interactome analysis

  • In situ protein interaction detection using proximity ligation assays

  • Split-HRP complementation for detecting specific HINFP-partner interactions

• Functional genomics approaches:

  • Integration with ATAC-seq data to correlate HINFP binding with chromatin accessibility

  • Combination with CUT&RUN or CUT&Tag for precise genomic localization

  • Correlation with histone modification patterns across the genome

These emerging applications represent the frontier of research utilizing HINFP antibodies, particularly those with enhanced properties through advanced conjugation methods.