PCMT1 Antibody

What is PCMT1 and what cellular functions does it regulate?

PCMT1 (protein-L-isoaspartate O-methyltransferase) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins . It functions by initiating repair of damaged proteins through methyl esterification of L-isoaspartyl and D-aspartyl residues, which are produced by spontaneous isomerization and racemization of L-aspartyl and L-asparaginyl residues in aging peptides and proteins .

PCMT1 acts on various proteins including EIF4EBP2, microtubule-associated protein 2, calreticulin, clathrin light chains, ubiquitin C-terminal hydrolase isozyme L1, phosphatidylethanolamine-binding protein 1, stathmin, beta-synuclein, and alpha-synuclein . Recent research has also revealed that PCMT1 can function as an instability factor in induced regulatory T cells by methylating the FOXP3 promoter . In oncology studies, PCMT1 has been identified as overexpressed in several tumors including prostate cancer, where it influences tumor progression and metastasis .

What are the recommended applications and dilutions for PCMT1 antibody?

Based on validated research protocols, PCMT1 antibody can be utilized in multiple applications with specific recommended dilutions for optimal results:

It's important to note that these dilutions serve as starting points, and researchers should optimize conditions for their specific experimental system . For antigen retrieval in IHC applications, TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may alternatively be used .

PCMT1 antibody has demonstrated positive Western blot detection in multiple sample types including HeLa cells, human testis tissue, HEK-293 cells, Raji cells, mouse testis tissue, and rat testis tissue. For immunohistochemistry, positive detection has been observed in human pancreas tissue, while immunofluorescence applications have shown successful detection in HEK-293 cells .

How should PCMT1 antibody be stored to maintain optimal activity?

For maximum stability and activity retention, PCMT1 antibody should be stored at -20°C, where it remains stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

For smaller quantities (such as 20μl sizes), the antibody may contain 0.1% BSA. Importantly, aliquoting is generally unnecessary for -20°C storage, simplifying laboratory workflows . When handling the antibody, minimize freeze-thaw cycles and keep on ice during experiments to preserve immunoreactivity.

How can I establish PCMT1 knockdown cell lines for functional studies?

To establish stable PCMT1 knockdown cell lines for functional studies, researchers can use lentiviral vectors carrying shRNAs targeting PCMT1. Based on published methodologies, the following protocol has proven effective:

• Generate lentiviral vectors carrying multiple shRNAs targeting PCMT1 (e.g., sh-PCMT1#1, sh-PCMT1#2, sh-PCMT1#3) and a control shRNA (sh-NC) .

• Infect target cells with the lentivirus for 48 hours .

• Select stable cell lines using puromycin. To determine the appropriate concentration:

  • Plate cells in a 6-well plate with different puromycin concentrations (0-15 μg/ml)

  • Incubate for 48 hours, changing the selective medium every 2 days

  • Evaluate cell survival rate using trypan blue staining every 2 days

  • Identify the minimum concentration that kills all non-transduced cells (typically 2 μg/ml)

• Maintain cells in this concentration of puromycin for selection.

• Verify knockdown efficiency using both qRT-PCR and Western blot analysis .

This approach allows for comprehensive functional studies of PCMT1's role in various cellular processes and disease mechanisms.

What is the recommended Western blot protocol for detecting PCMT1 protein?

For optimal detection of PCMT1 protein via Western blot, the following methodology is recommended based on published research protocols:

• Protein Extraction:

  • Isolate total protein from cells using RIPA lysis buffer according to manufacturer's instructions .

• Protein Separation:

  • Load approximately 50 μg of protein lysate on 12% SDS-PAGE gels .

  • Transfer proteins onto 0.45 μm polyvinylidene difluoride (PVDF) membranes.

• Antibody Incubation:

  • Block membranes with appropriate blocking buffer.

  • Incubate overnight with anti-PCMT1 primary antibody (1:1000 dilution, catalog #10519-1-AP) .

  • Use β-actin (1:1000 dilution, catalog #20536-1-AP) as loading control .

  • Incubate with HRP-conjugated secondary antibodies (1:3000 dilution) .

• Detection:

  • Perform detection using ECL (enhanced chemiluminescence) substrate .

  • Observe PCMT1 at the expected molecular weight of 25-28 kDa .

This protocol consistently yields clear and specific detection of PCMT1 protein across various cell types and tissues.

How can I quantify PCMT1 mRNA expression in experimental samples?

For accurate quantification of PCMT1 mRNA expression, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the recommended method. The following protocol outlines the key steps:

• RNA Extraction:

  • Extract total RNA from cell lines or tissue samples using an appropriate RNA isolation kit.

  • Assess RNA quality and quantity via spectrophotometry.

• cDNA Synthesis:

  • Perform reverse transcription to generate cDNA from extracted RNA.

  • Use equal amounts of RNA input for all samples to ensure comparability.

• qPCR Amplification:

  • Design specific primers for PCMT1 and suitable reference genes.

  • Perform qPCR reactions in technical triplicates.

• Data Analysis:

  • Calculate the relative expression of PCMT1 mRNA using the 2^-ΔΔCt method .

  • Normalize to stable reference genes.

  • Analyze expression data using statistical software such as GraphPad Prism 8 .

This methodology enables reliable quantification of PCMT1 expression changes in response to experimental manipulations or between different sample types.

What is the significance of PCMT1 expression in cancer progression?

PCMT1 has emerged as a significant player in cancer biology, with multiple studies demonstrating its relevance to cancer progression. In prostate cancer (PCa), PCMT1 is significantly overexpressed in cancer tissues compared to normal prostate or benign prostatic hyperplasia (BPH) tissues . Analysis of The Cancer Genome Atlas Program (TCGA) database has confirmed this upregulation .

The clinical significance of PCMT1 expression is evidenced by its correlation with several critical clinical parameters:

PCMT1 expression was not significantly associated with patient age (P = 0.053) or preoperative prostate-specific antigen (PSA) levels (P = 0.679) .

Functional studies have demonstrated that PCMT1 inhibition significantly suppresses the proliferation, migration, and invasion of PCa cells while promoting apoptosis . In vivo tumor formation experiments in nude mice have further confirmed that PCMT1 promotes tumor growth, suggesting its crucial role in facilitating cancer progression .

How does PCMT1 function as a pan-cancer immune biomarker?

Recent research has established PCMT1 as a significant pan-cancer immune biomarker with implications for immune checkpoint inhibitor therapies (ICIs). Both genetic variations and methylation of PCMT1 significantly impact its expression levels across different cancer types .

PCMT1 expression correlates with several critical survival parameters:

The immunological significance of PCMT1 is evidenced by its correlation with immune cell infiltration patterns:

• PCMT1 expression shows a significant positive correlation with CD8+ T cell infiltration in 14 different malignancies .

• In kidney renal clear cell carcinoma (KIRC) and liver hepatocellular carcinoma (LIHC), PCMT1 expression positively correlates with infiltration levels of all six major immunological cell types analyzed .

• PCMT1 expression shows significant associations with stromal scores, microenvironment metrics, and immune scores in most tumor types .

• Specific immune cell correlations include:

  • Positive correlation with CD4+ T helper 2 (Th2) cells

  • Negative correlation with central and effector memory T cells, CD8+ memory T cells, and CD4+ T helper 1 (Th1) cells in most tumors

These findings suggest PCMT1 may serve as a valuable biomarker for predicting immunotherapy responses and understanding the tumor immune microenvironment.

What methodologies are recommended for analyzing PCMT1's role in tumor immunity?

For comprehensive analysis of PCMT1's role in tumor immunity, researchers should employ a multi-faceted approach combining bioinformatic and experimental methodologies:

• Correlation Analysis with Immune Infiltrates:

  • Utilize the "immunedeconv" R package to correlate PCMT1 expression with levels of immune cell infiltrates .

  • Calculate correlation coefficients using the Spearman method for non-parametric assessment .

• Association with Immune Checkpoint Markers:

  • Assess correlations between PCMT1 expression and established immune checkpoints including:

    • CD274 (PD-L1), SIGLEC15, CTLA-4, HAVCR2, PDCD1 (PD-1), LAG3, PDCD1LG2, and TIGIT .

• Biomarker Analysis for Immunotherapy Response:

  • Evaluate correlations between PCMT1 and established biomarkers of immunotherapy response:

    • Tumor Mutational Burden (TMB)

    • Microsatellite Instability (MSI) .

• Single Sample Gene Set Enrichment Analysis (ssGSEA):

  • Implement ssGSEA through the GSVA package to analyze immune infiltration patterns .

  • Classify and analyze 24 distinct immune cell types in relation to PCMT1 expression .

• Statistical Analysis:

  • Use Spearman's correlation and Wilcoxon rank-sum tests to assess relationships between PCMT1 and immune parameters .

  • Consider p-values <0.05 as statistically significant .

These methodologies provide a comprehensive framework for investigating PCMT1's immunological functions in cancer and may guide the development of immunotherapeutic strategies targeting PCMT1-related pathways.

How can PCMT1 be targeted for therapeutic applications in cancer and immune disorders?

Emerging research suggests several promising approaches for targeting PCMT1 therapeutically:

• Cell-Penetrating Antibody Technology:
  Research has demonstrated that targeting PCMT1 using a cell-penetrating antibody can effectively modulate RNA processing to confer a stable regulatory T cell phenotype . This approach involves using synthetic cell-penetrating peptide mimics for intracellular anti-protein kinase C theta (PKCθ) delivery, which in turn affects PCMT1 activity . The mechanism appears to involve PCMT1's role as an instability factor in induced regulatory T cells through methylation of the FOXP3 promoter .

• RNA Interference (RNAi) Approaches:
  Knockdown of PCMT1 using siRNAs has been shown to significantly suppress the proliferation, migration, and invasion of cancer cells while promoting apoptosis . This suggests that RNAi-based therapeutic approaches targeting PCMT1 could have anti-tumor effects in cancers where PCMT1 is overexpressed.

• Targeting PCMT1-Dependent Immune Modulation:
  Given PCMT1's significant correlations with immune cell infiltration and immune checkpoint molecules across multiple cancer types , targeting PCMT1 could potentially enhance immunotherapy responses. This might be particularly relevant in tumors where PCMT1 expression correlates with altered T cell functionality or immune checkpoint expression.

For effective development of these therapeutic strategies, researchers should consider the tissue-specific roles of PCMT1 and potential off-target effects, as PCMT1 is involved in normal protein repair mechanisms in healthy tissues.

What are common technical challenges in PCMT1 antibody-based experiments and how can they be addressed?

Researchers working with PCMT1 antibodies may encounter several technical challenges that can influence experimental outcomes. Here are common issues and recommended solutions:

• Non-specific Binding:

  • Challenge: PCMT1 antibodies may occasionally show cross-reactivity with other proteins.

  • Solution:

    • Validate antibody specificity using PCMT1 knockdown or knockout controls .

    • Optimize blocking conditions using 3-5% BSA or milk in TBST.

    • Include appropriate negative controls in all experiments.

• Variable Detection in Different Sample Types:

  • Challenge: Detection sensitivity may vary between human, mouse, and rat samples.

  • Solution:

    • Adjust antibody concentrations based on species (PCMT1 antibody 10519-1-AP shows reactivity with human, mouse, and rat samples) .

    • Optimize extraction protocols to ensure sufficient protein recovery from different tissue types.

• Inconsistent Immunohistochemistry Results:

  • Challenge: Antigen retrieval efficacy can significantly impact IHC staining quality.

  • Solution:

    • For optimal results, use TE buffer pH 9.0 for antigen retrieval as recommended .

    • As an alternative, citrate buffer pH 6.0 may be used .

    • Test multiple antigen retrieval conditions if initial results are suboptimal.

• Quantification Challenges in Western Blot:

  • Challenge: The observed molecular weight of PCMT1 (25-28 kDa) may show slight variations .

  • Solution:

    • Use appropriate molecular weight markers spanning the 20-30 kDa range.

    • Consider using gradient gels for better resolution in this molecular weight range.

    • Ensure complete denaturation of samples before loading.

By implementing these technical solutions, researchers can enhance the reliability and reproducibility of PCMT1 antibody-based experiments across various applications.

How can PCMT1 expression data be integrated with clinical parameters for prognostic modeling?

Integration of PCMT1 expression data with clinical parameters enables development of sophisticated prognostic models with potential clinical utility. The following methodological framework is recommended:

• Data Collection and Preprocessing:

  • Obtain RNA-seq and clinical data from reliable databases such as TCGA .

  • Normalize expression data and handle missing values appropriately.

  • Categorize continuous variables as needed for statistical analysis.

• Statistical Modeling Approach:

  • Perform univariate and multivariate Cox regression analyses to determine if PCMT1 serves as an independent predictive factor .

  • Evaluate the diagnostic value of PCMT1 using receiver operating characteristic (ROC) curves, which can be generated with the "pROC" and "ggplot2" packages in R .

• Nomogram Development:

  • Create a comprehensive nomogram incorporating PCMT1 expression with relevant clinicopathological features .

  • This visual predictive tool can estimate individual patient outcomes based on PCMT1 expression and other parameters.

• Integration with Immune Parameters:

  • Since PCMT1 functions as an immune biomarker, incorporate immune infiltration analysis using methods such as ssGSEA .

  • Correlate PCMT1 expression with levels of various immune cell types using Spearman's correlation technique and the Wilcoxon rank-sum test .

  • Consider p-values <0.05 as statistically significant for correlations .

• Validation Strategies:

  • Implement both internal validation (e.g., bootstrapping) and external validation using independent cohorts.

  • Assess model performance using concordance index (C-index), calibration plots, and decision curve analysis.

This integrated approach allows researchers to develop robust prognostic models that combine PCMT1 expression with clinical parameters, potentially improving risk stratification and treatment decision-making for cancer patients.

What are the most promising future research directions for PCMT1 antibody applications?

PCMT1 antibody research appears poised for several high-impact future directions:

• Therapeutic Applications in Cancer:
  Given PCMT1's demonstrated role in cancer progression and its correlation with clinical parameters such as tumor stage and metastasis, developing therapeutic antibodies targeting PCMT1 represents a promising avenue. Cell-penetrating antibody technology has already shown potential in modulating PCMT1 activity in regulatory T cells , suggesting similar approaches might be effective against PCMT1 in cancer cells.

• Immunotherapy Biomarker Development:
  The established correlations between PCMT1 expression and immune cell infiltration across multiple cancer types suggest PCMT1 could serve as a predictive biomarker for immunotherapy response. Future research should investigate whether PCMT1 expression levels can predict response to immune checkpoint inhibitors and other immunotherapeutic approaches.

• Multiplex Imaging Applications:
  Development of multiplex immunofluorescence protocols incorporating PCMT1 antibodies could enable spatial analysis of PCMT1 in relation to immune cell populations within the tumor microenvironment, providing deeper insights into its mechanistic roles in cancer immunity.

• Post-Translational Modification Analysis:
  Since PCMT1 itself is involved in protein repair through modification of damaged residues, investigating how PCMT1 is regulated through its own post-translational modifications could reveal novel regulatory mechanisms and therapeutic targets.

These research directions would significantly advance our understanding of PCMT1's roles in normal physiology and disease, potentially leading to new diagnostic and therapeutic applications.

How can researchers address contradictory findings in PCMT1 research literature?

When encountering contradictory findings regarding PCMT1 function or expression across different studies, researchers should implement the following systematic approach:

• Contextual Analysis:

  • Consider tissue-specific differences in PCMT1 function. PCMT1 shows different expression patterns and associations across cancer types , suggesting context-dependent roles.

  • Evaluate methodological differences between studies, including antibody clones, detection methods, and experimental conditions.

• Isoform-Specific Analysis:

  • Determine whether studies are examining the same PCMT1 isoforms or splice variants.

  • Design experiments to specifically detect and analyze individual PCMT1 isoforms.

• Integration of Multiple Analytical Approaches:

  • Combine protein-level (Western blot, IHC) and transcript-level (qRT-PCR, RNA-seq) analyses to obtain a more comprehensive view of PCMT1 expression and function.

  • Supplement with functional studies using knockdown/overexpression approaches .

• Consideration of Technical Variables:

  • Standardize antibody validation procedures using positive and negative controls.

  • Report detailed methodological parameters including antibody dilutions, incubation conditions, and detection methods .

• Meta-analysis Approach:

  • When sufficient data are available, conduct meta-analyses of PCMT1 expression or functional studies across different experimental systems or cancer types.

  • Weight findings based on sample size, methodological rigor, and consistency of results.