ANKRD42 Antibody,HRP conjugated

What is ANKRD42 and why is it studied in molecular research?

ANKRD42 (Ankyrin repeat domain 42) is a human protein of approximately 43 kDa molecular weight that contains 389 amino acids . It's also known by alternative names including SARP and PPP1R79 . Despite ongoing research, the specific function of this protein remains largely unknown, making it an interesting target for fundamental research into protein interactions and cellular functions . The protein is known to interact with PPP1CC and UBC, suggesting potential roles in cellular signaling pathways or protein regulation . Research on ANKRD42 typically involves detecting its expression patterns across different tissues and investigating its potential involvement in cellular processes through antibody-based techniques.

What is HRP conjugation and why is it valuable for antibody applications?

Horseradish peroxidase (HRP) conjugation is a laboratory technique where the enzyme HRP (a 44 kDa glycoprotein) is chemically attached to antibodies to create detection reagents for various immunoassay applications . This conjugation is valuable because HRP catalyzes reactions that produce colorimetric, chemiluminescent, or fluorescent signals when exposed to appropriate substrates . These signals can be precisely measured, allowing for sensitive detection of target proteins.

The primary advantages of HRP-conjugated antibodies include:

• High sensitivity for detecting low-abundance proteins

• Excellent performance in immunoblotting and ELISA applications

• Capability for visualization through chromogenic reactions (e.g., converting diaminobenzidine into a brown pigment in the presence of hydrogen peroxide)

• Compatibility with multiple substrate options including ABTS, TMB, and TMBUS

• Option for direct detection (conjugated to primary antibody) or indirect detection (conjugated to secondary antibody)

What are the available formats of anti-ANKRD42 antibodies?

Based on available research resources, anti-ANKRD42 antibodies are primarily found in the following formats:

• Rabbit polyclonal antibodies against human ANKRD42 at 0.2 mg/ml concentration

• Rabbit polyclonal antibodies targeting the N-terminal region of human ANKRD42 at 0.5 mg/ml concentration

These antibodies are typically supplied in liquid format purified in 1x PBS buffer with additives such as 0.09% (w/v) sodium azide and 2% sucrose to maintain stability . The immunogen used for raising these antibodies is usually a synthetic peptide directed towards the N-terminal region of human ANKRD42, with the specific sequence: MPGVANSGPSTSSRETANPCSRKKVHFGSIHDAVRAGDVKQLSEIVCLHW .

How is an ANKRD42 antibody conjugated to HRP, and what protocols yield optimal results?

The conjugation of ANKRD42 antibodies to HRP follows similar protocols to other antibody-HRP conjugations. The process typically involves:

• Periodate Oxidation Method: Sodium meta-periodate is used to oxidize the carbohydrate moieties on the HRP molecule, generating reactive aldehyde groups . These aldehydes can then form covalent bonds with amine groups on the antibody.

• Lightning-Link® Approach: For researchers seeking simplified methods, Lightning-Link® HRP systems allow direct conjugation of antibodies to HRP without extensive laboratory procedures . This method is advantageous for maintaining antibody performance while reducing labor.

Key considerations for optimal ANKRD42 antibody-HRP conjugation include:

• Buffer composition is critical for successful conjugation; common buffer additives can impair the process

• The antibody:HRP ratio should be optimized to maintain both antibody binding capacity and enzymatic activity

• Stabilizing agents such as LifeXtend™ HRP conjugate stabilizer can protect antibody-HRP conjugates from performance degradation

• Lyophilization (freeze-drying) has been shown to enhance HRP-antibody conjugation in some contexts

The recommended protocol involves careful purification of the antibody, activation of HRP with periodate, mixing at an optimized ratio, and inclusion of appropriate stabilizers to ensure long-term activity of the conjugate.

What are the validated applications for ANKRD42 antibodies and how can HRP conjugation enhance these applications?

ANKRD42 antibodies have been validated for several key applications:

• Western Blotting (WB): Primary application for detecting ANKRD42 protein in cell or tissue lysates

• Immunohistochemistry (IHC): For localization of ANKRD42 in tissue sections

• Immunocytochemistry-Immunofluorescence (ICC-IF): For cellular localization studies

HRP conjugation can enhance these applications in several ways:

• Direct Detection: HRP-conjugated primary antibodies against ANKRD42 would allow one-step detection, reducing cross-species reactivity issues and eliminating additional wash steps .

• Enhanced Sensitivity: HRP conjugation offers "unparalleled sensitivity and performance" for detecting low-abundance proteins like ANKRD42 .

• Versatility in Readout Methods: Researchers can choose between chemiluminescent, colorimetric, or other detection methods depending on their specific experimental requirements .

• Time Efficiency: Direct conjugates eliminate the need for secondary antibody incubation, shortening experimental protocols .

What dilution and experimental conditions are optimal for using HRP-conjugated antibodies in ANKRD42 detection?

For optimal detection of ANKRD42 using HRP-conjugated antibodies, the following conditions are recommended:

Western Blotting Applications:

• Typical dilution range: 1:1000 (similar to other HRP-conjugated antibodies)

• Blocking solution: 5% non-fat milk or BSA in TBST

• Incubation temperature: 4°C overnight or room temperature for 1-2 hours

• Washing buffer: TBST (Tris-buffered saline with 0.1% Tween-20)

• Recommended substrate: Works optimally with chemiluminescent substrates like Azure Radiance

Storage Conditions:

• Short-term storage (up to 1 week): 2-8°C

• Long-term storage: -20°C in small aliquots to prevent freeze-thaw cycles

• Important note: Many HRP-conjugated antibodies should not be aliquoted to maintain stability

For ANKRD42 detection specifically, it's important to note the protein's molecular weight (approximately 43kDa) when interpreting Western blot results .

What are common challenges in using HRP-conjugated antibodies for ANKRD42 detection, and how can they be addressed?

Researchers working with HRP-conjugated antibodies for ANKRD42 detection may encounter several challenges:

Challenge 1: Antibody Specificity Issues

• Problem: Cross-reactivity with other ankyrin repeat-containing proteins.

• Solution: Verify antibody specificity using knockout/knockdown controls or competitive binding assays with the immunizing peptide sequence (MPGVANSGPSTSSRETANPCSRKKVHFGSIHDAVRAGDVKQLSEIVCLHW) .

Challenge 2: Weak Signal or High Background

• Problem: Insufficient detection sensitivity or non-specific binding.

• Solution: Optimize antibody concentration, incubation time/temperature, and blocking conditions. Consider using enhanced chemiluminescent substrates specifically designed for HRP detection . The Azure Radiance chemiluminescent substrates are optimized to work with HRP-conjugated secondary antibodies .

Challenge 3: Conjugate Stability Issues

• Problem: Loss of enzymatic activity during storage or use.

• Solution: Use stabilizing agents like LifeXtend™ HRP conjugate stabilizer . Store properly at -20°C in small aliquots to prevent freeze-thaw cycles . Some HRP-conjugated antibodies should not be aliquoted at all .

Challenge 4: Buffer Compatibility Issues

• Problem: Common buffer additives can interfere with conjugation and performance.

• Solution: Ensure antibody preparation is free of interfering agents before conjugation, or use commercial Lightning-Link® HRP systems designed to overcome buffer compatibility issues .

How can ANKRD42 antibody, HRP conjugated be used in multiplex immunoassays?

Advanced multiplex immunoassays involving HRP-conjugated ANKRD42 antibodies require careful experimental design:

Methodological Approach:

• Sequential Detection: If using multiple HRP-conjugated antibodies, implement sequential detection with thorough stripping between rounds using appropriate HRP stripping buffer .

• Differential Labeling Strategy: Combine HRP-conjugated ANKRD42 antibody with antibodies conjugated to different reporter enzymes (like alkaline phosphatase) or fluorophores for simultaneous detection of multiple targets.

• Species Selection: When designing multiplex experiments, select primary antibodies from different host species (e.g., rabbit anti-ANKRD42 with mouse anti-target2) and use species-specific secondary antibodies conjugated to different detection systems.

Technical Considerations:

• Evaluate potential cross-reactivity between antibodies before multiplexing

• Validate signal specificity using appropriate controls

• Optimize individual antibody concentrations to achieve balanced signal strength across targets

• Consider signal development timing when using multiple chromogenic substrates

What advanced validation techniques are recommended to confirm ANKRD42 antibody specificity and performance after HRP conjugation?

For rigorous research applications, validation of HRP-conjugated ANKRD42 antibodies should include:

Validation Technique 1: Peptide Competition Assay

• Pre-incubate the HRP-conjugated antibody with excess blocking peptide (sequence: MPGVANSGPSTSSRETANPCSRKKVHFGSIHDAVRAGDVKQLSEIVCLHW)

• Run parallel assays with blocked and unblocked antibody

• Specific signals should disappear in the blocked condition

Validation Technique 2: Genetic Knockdown/Knockout Controls

• Test antibody on samples from ANKRD42 knockdown/knockout models

• Specific signals should be reduced or eliminated in these models

Validation Technique 3: Multi-platform Confirmation

• Compare results across different applications (WB, IHC, ICC-IF)

• Consistent patterns across platforms strengthen confidence in specificity

Validation Technique 4: Enzymatic Activity Assessment

• Evaluate HRP enzymatic activity using standard substrates like TMB

• Compare signal intensity to non-conjugated antibody + HRP-conjugated secondary antibody system

• Ensure conjugation hasn't compromised detection sensitivity

How should researchers interpret Western blot results when using HRP-conjugated ANKRD42 antibodies?

When analyzing Western blot data using HRP-conjugated ANKRD42 antibodies, researchers should consider:

Expected Molecular Weight and Banding Pattern:

• ANKRD42 has a molecular weight of approximately 43 kDa

• Evaluate whether observed bands match the expected molecular weight

• Be aware that post-translational modifications may cause shifts in apparent molecular weight

Signal Specificity Assessment:

• Compare bands against appropriate positive and negative controls

• Validate using genetic manipulation (knockdown/overexpression) when possible

• Consider the predicted species cross-reactivity based on immunogen sequence homology:

  • 100% homology: Human, Cow, Pig, Rabbit

  • 93% homology: Mouse, Rat, Dog

  • 86% homology: Horse

  • 79% homology: Guinea Pig

Quantitative Analysis Considerations:

• Use appropriate normalization with housekeeping proteins

• Implement densitometric analysis using standard software

• Ensure the chemiluminescent signal falls within the linear range of detection

• Consider the high sensitivity of HRP-conjugated antibodies when determining optimal exposure times

What are the expected expression patterns of ANKRD42 across different tissues and experimental systems?

Although detailed expression data for ANKRD42 is limited in the provided search results, researchers can anticipate:

Tissue Expression Patterns:

• As ANKRD42 antibodies are validated for human samples, primary analysis should focus on human cell lines and tissues

• Based on sequence homology, the antibodies may cross-react with ANKRD42 from multiple species including mouse, rat, cow, dog, guinea pig, horse, pig, and rabbit

Cellular Localization:

• The provided search results don't specify subcellular localization of ANKRD42

• Researchers should use ICC-IF with appropriate subcellular markers to determine localization

Experimental Systems:

• Western blotting is the primary validated application

• Researchers may need to optimize conditions for other applications

When analyzing ANKRD42 expression data, researchers should compare their findings with publicly available transcriptomic and proteomic databases to contextualize results within broader expression patterns.

How can researchers quantitatively compare ANKRD42 expression between experimental conditions using HRP-conjugated antibodies?

For quantitative comparative analysis of ANKRD42 expression using HRP-conjugated antibodies:

Methodological Approach:

• Standardization of Protein Loading:

  • Use consistent total protein loading (validated by stain-free technology or housekeeping proteins)

  • Consider BCA or Bradford assays prior to gel loading for accurate quantification

• Signal Acquisition Optimization:

  • Capture images using a dynamic range detection system

  • Ensure exposure times avoid signal saturation

  • For chemiluminescent detection, capture multiple exposures to ensure linearity

• Densitometric Analysis Protocol:

  • Use specialized software (ImageJ, Image Studio, etc.) for band intensity quantification

  • Subtract background signal appropriately

  • Normalize ANKRD42 signal to loading controls

• Statistical Analysis:

  • Perform experiments with appropriate biological and technical replicates (minimum n=3)

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Present data with error bars representing standard deviation or standard error

Technical Considerations:

• The high sensitivity of HRP detection may require careful titration of antibody dilutions to enable accurate quantitation

• When comparing across multiple blots, include a common reference sample on each blot for inter-blot normalization

• Consider the impact of detection substrate selection on signal-to-noise ratio and dynamic range

By following these methodological approaches, researchers can obtain reliable quantitative data regarding ANKRD42 expression changes across different experimental conditions.