HRSP12 Antibody

What is HRSP12 and what cellular functions does it perform?

HRSP12 (heat-responsive protein 12) is a ribonuclease found predominantly in human adult kidney and liver tissues. It functions primarily by inhibiting translation through the cleavage of mRNA molecules. The protein specifically cleaves phosphodiester bonds in single-stranded RNA, suggesting its role in RNA metabolism and potentially in gene expression regulation . HRSP12 has a calculated molecular weight of approximately 14.5 kDa, which matches its observed molecular weight in experimental settings .

What are the primary applications for HRSP12 antibodies in research?

HRSP12 antibodies are primarily used in Western Blot (WB) and Immunohistochemistry (IHC) applications. For Western blotting, the recommended dilution typically ranges from 1:500 to 1:2000, while for immunohistochemistry, dilutions between 1:20 and 1:200 are commonly used . These applications allow researchers to detect and quantify HRSP12 protein expression in various tissue samples and cell lines, particularly useful in studies comparing normal versus pathological states.

What tissue types show significant HRSP12 expression?

HRSP12 expression is most prominent in human adult kidney and liver tissues . Interestingly, the levels of both HRSP12 mRNA and protein are markedly reduced in hepatocellular tumors and human hepatoma cell lines compared to normal liver tissues . This differential expression pattern makes HRSP12 a potential marker for liver pathology studies and cancer research.

What species reactivity can be expected from commercial HRSP12 antibodies?

Commercial HRSP12 antibodies typically show reactivity with human and rat samples . The antibodies available from major suppliers such as Sigma Aldrich and Proteintech have been tested and validated for these species. When working with other species, researchers should conduct preliminary validation tests to confirm cross-reactivity before proceeding with full experiments.

How should HRSP12 antibodies be validated for specific research applications?

According to the International Working Group of Antibody Validation (IWGAV), researchers should implement at least one of their five recommended validation pillars :

• Genetic strategies: Testing the antibody in conditions when HRSP12 is not expressed (e.g., knockout models or siRNA knockdown)

• Orthogonal strategies: Comparing antibody results with HRSP12 levels measured by other techniques

• Independent antibody strategy: Comparing results with alternative HRSP12 antibodies targeting different epitopes

• Expression of tagged proteins: Using affinity tags or fluorescent protein-tagged HRSP12

• Immunocapture followed by mass spectrometry: Confirming the identity of the captured protein

For HRSP12 specifically, validation data available from suppliers like Sigma Aldrich indicates their antibodies have undergone independent and orthogonal RNAseq validation , providing higher confidence in specificity.

What are the cellular localization patterns of HRSP12 and how can this affect experimental design?

HRSP12 is predominantly expressed in the cytoplasm, though occasionally it can be detected in the nucleus of less differentiated cells . This dual localization pattern has important implications for experimental design:

When designing immunofluorescence or subcellular fractionation experiments, researchers should account for this variable localization, particularly when studying cell differentiation or cancer models.

How does HRSP12 expression change in pathological conditions?

Studies have shown that HRSP12 levels (both mRNA and protein) are significantly reduced in hepatocellular tumors and human hepatoma cell lines compared to normal liver tissues . This finding suggests HRSP12 may have tumor suppressor properties or serve as a differentiation marker in liver tissue. When investigating liver pathologies, researchers should consider:

• Comparing HRSP12 expression between normal and diseased tissues

• Correlating HRSP12 levels with disease progression markers

• Investigating the mechanistic relationship between HRSP12 downregulation and disease pathogenesis

These analyses may provide insights into the role of HRSP12 in liver pathophysiology and potentially identify new diagnostic or therapeutic targets.

What are the optimal immunohistochemistry conditions for HRSP12 detection?

For optimal HRSP12 detection in immunohistochemistry applications, consider the following protocol recommendations:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results; alternatively, citrate buffer pH 6.0 can be used with potentially different outcomes

• Antibody dilution: Typically 1:20-1:200, but should be optimized for each specific tissue type and experimental condition

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

• Detection system: HRP-conjugated secondary antibodies with DAB or fluorescent-labeled secondaries are both suitable

When optimizing your protocol, remember that HRSP12 shows differential expression between normal and cancerous tissues, which may affect staining intensity and pattern interpretation.

What are the recommended Western blotting conditions for HRSP12 detection?

For Western blot applications detecting HRSP12:

• Sample preparation: Standard RIPA or NP-40 lysis buffers are suitable

• Protein loading: 20-40 μg of total protein per lane is typically sufficient

• Gel percentage: 12-15% polyacrylamide gels are recommended for optimal resolution of the 14.5 kDa HRSP12 protein

• Transfer conditions: Semi-dry or wet transfer systems work well; use PVDF membranes for best results

• Antibody concentration: Use at 0.04-0.4 μg/mL or dilutions of 1:500-1:2000

• Expected band size: 14.5 kDa

When validating Western blot results, always include positive control samples known to express HRSP12 (e.g., normal human liver tissue lysates).

What controls should be included in experiments using HRSP12 antibodies?

To ensure experimental validity when working with HRSP12 antibodies, include these controls:

• Positive tissue controls: Human liver or kidney tissue lysates for Western blot; normal liver sections for IHC

• Negative controls:

  • Omission of primary antibody

  • Pre-adsorption controls (though these have limitations in specificity determination)

  • Tissues known to express minimal HRSP12

• Validation controls:

  • HRSP12 knockdown or knockout samples (when available)

  • Independent detection with alternative antibodies targeting different epitopes

• Loading/processing controls: GAPDH, β-actin, or tubulin for Western blot; tissue architecture verification for IHC

These controls help distinguish between specific signal and background, ensuring reliable interpretation of experimental results.

What are common issues with HRSP12 Western blot detection and their solutions?

When encountering problems with HRSP12 Western blot experiments, consider these common issues and solutions:

For HRSP12 specifically, ensure you're using reducing conditions in sample preparation to observe the expected 14.5 kDa band.

How can researchers address non-specific binding in HRSP12 immunohistochemistry?

Non-specific binding in HRSP12 immunohistochemistry can be minimized through these strategies:

• Optimize blocking conditions:

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature)

  • Consider adding 0.1-0.3% Triton X-100 for better penetration

• Antibody optimization:

  • Test multiple dilutions to find optimal concentration

  • Increase washing steps (5-6 washes of 5 minutes each)

  • Consider longer but more dilute antibody incubations

• Tissue preparation improvements:

  • Ensure proper fixation (overfixation can cause artifactual staining)

  • Test multiple antigen retrieval methods

  • Consider freshly cut sections versus stored slides

• Validation approaches:

  • Perform peptide competition assays

  • Compare staining patterns with published HRSP12 expression data

How should researchers quantify HRSP12 expression in immunohistochemistry?

Quantification of HRSP12 expression in immunohistochemistry can be approached through several methods:

• Scoring systems:

  • H-score: Combines intensity (0-3) and percentage of positive cells

  • Allred score: Sum of proportion score (0-5) and intensity score (0-3)

  • Simple percentage scoring: Estimate of positive cells as percentage

• Digital image analysis:

  • Use software like ImageJ, QuPath, or commercial platforms

  • Set consistent thresholds across all analyzed samples

  • Consider separate analysis of nuclear versus cytoplasmic staining

• Reporting standards:

  • Always include representative images

  • Report scoring method in detail

  • Include information on antibody validation

  • Document observer blinding procedures if applicable

When comparing normal versus pathological tissues, matched pairs from the same patient provide the most reliable comparative data.

How can researchers correlate HRSP12 protein levels with mRNA expression data?

Correlating HRSP12 protein levels with mRNA expression requires careful consideration of several factors:

• Sample preparation:

  • Use parallel samples for protein and RNA extraction

  • Consider laser capture microdissection for tissue heterogeneity

• Quantification methods:

  • For protein: Western blot densitometry or quantitative IHC

  • For mRNA: qRT-PCR, RNA-seq, or microarray data

• Data normalization:

  • Normalize protein expression to appropriate housekeeping proteins

  • Normalize mRNA to stable reference genes

  • Consider using multiple reference controls

• Statistical analysis:

  • Calculate Pearson or Spearman correlation coefficients

  • Use linear regression models

  • Consider time-course analyses if regulation mechanisms are being studied

Remember that HRSP12's mRNA and protein levels are both reported to be reduced in hepatocellular tumors compared to normal liver tissue , suggesting coordinated regulation at both levels.

What is the relationship between RNA modification pathways and applications of HRSP12 antibodies?

Recent research into RNA modification mechanisms provides context for HRSP12 studies. While not directly addressing HRSP12, studies of N6-methyladenosine (m6A) modifications and their effect on antibody production highlight the importance of RNA regulatory mechanisms . As HRSP12 functions as a ribonuclease affecting translation, researchers should consider:

• Potential interactions between HRSP12 and modified RNA substrates

• Whether HRSP12 shows preferential activity toward specific RNA modifications

• The role of HRSP12 in post-transcriptional regulation pathways

These considerations may open new research directions for understanding HRSP12's biological role beyond its currently established functions.

How do antibody validation standards affect HRSP12 antibody research reliability?

The scientific community has recognized significant issues with antibody validation that directly impact research using HRSP12 antibodies. Studies have shown that approximately half of commercial antibodies may not be suitable for their intended applications . To ensure reliability in HRSP12 research:

• Follow published validation guidelines:

  • Implement at least one of the IWGAV validation pillars

  • Document validation results in publications

• Report detailed antibody information:

  • Include catalogue and batch numbers

  • Report RRID (Research Resource Identifier)

• Perform application-specific validation:

  • Validate antibodies for each specific technique

  • Re-validate new lots/batches of antibodies

• Share validation data:

  • Contribute to antibody validation databases

  • Include supplementary validation data with publications

By adhering to these standards, researchers can significantly improve the reliability and reproducibility of HRSP12 antibody-based experiments.