PRMT1 Antibody

What is PRMT1 and what cellular functions does it regulate?

PRMT1 (Protein Arginine Methyltransferase 1) is a major type I arginine methyltransferase that catalyzes asymmetric dimethylation of arginine residues on substrate proteins. It plays critical roles in various cellular processes including:

• Transcriptional regulation through histone modifications (primarily H4R3me2a)

• Metabolism regulation, particularly glycolysis

• RNA processing and transport

• DNA damage response

• Cell signaling pathway modulation (EGFR, Wnt)

• Cell proliferation and survival

PRMT1 is highly expressed across multiple tissues and acts as the predominant type I PRMT in mammalian cells, accounting for approximately 75% of cellular arginine methylation activity .

In which diseases is PRMT1 implicated as a potential biomarker or therapeutic target?

Based on current research, PRMT1 has been implicated in several pathological conditions:

• Cancer progression: Overexpressed in colorectal cancer (CRC), breast cancer (including triple-negative breast cancer), lung cancer, and leukemia

• Immune disorders: Regulates B cell activation, germinal center formation, and T-cell responses

• Male infertility: Critical for spermatogonial homeostasis

• Systemic sclerosis: Identified as a potential autoantibody target

The table below summarizes PRMT1's role in different cancer types:

How do I select the appropriate PRMT1 antibody for my specific application?

When selecting a PRMT1 antibody, consider the following criteria based on your experimental requirements:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, IP, or FC). For example, Proteintech's 11279-1-AP has been validated for WB, IHC, IF/ICC, FC, and IP, while Cell Signaling's F339 antibody (#2453) is only validated for WB .

• Species reactivity: Confirm reactivity with your model organism. Most commercial PRMT1 antibodies react with human, mouse, and rat PRMT1, but verify cross-reactivity if working with other species.

• Epitope specificity: Consider which region of PRMT1 the antibody recognizes:

  • N-terminal antibodies may detect all PRMT1 isoforms

  • C-terminal antibodies might miss truncated variants

  • Some antibodies recognize specific post-translational modifications

• Published validation: Review literature citations that have used the antibody successfully in applications similar to yours.

• Dilution recommendations: Check the manufacturer's recommended dilutions for your application. For example, Proteintech's 11279-1-AP recommends 1:2000-1:16000 for WB and 1:50-1:500 for IHC .

What controls should I include when validating a PRMT1 antibody?

Proper validation requires several controls:

• Positive controls: Use cell lines or tissues known to express high levels of PRMT1. Based on the search results, these include:

  • A549, MCF-7, HeLa, Jurkat, and HepG2 cells

  • Mouse/rat brain tissue

  • Spermatogonial populations in testicular tissue

  • Germinal center B cells after immunization

• Negative controls:

  • PRMT1 knockout or knockdown samples (PRMT1-null B cells, PRMT1-sKO testicular lysates)

  • Irrelevant IgG matched to the PRMT1 antibody's host species

  • Peptide competition/blocking with the immunizing peptide

• Specificity controls:

  • Secondary antibody-only controls to assess background

  • Tissue samples from conditional knockout models (like Prmt1^f/f CD23Cre mice)

  • Validation in multiple applications (e.g., if WB shows a single band at 40-42 kDa, verify with IP or IF)

Example validation workflow from literature: "To confirm the validity of antigen capture process, topoisomerase I (Topo I) was detected by western blotting following IP in a patient who was positive for anti-Topo I antibody (ATA), whereas undetectable in an ATA-negative patient" .

What are the optimal conditions for using PRMT1 antibodies in Western blotting?

For optimal Western blot results with PRMT1 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 lysis buffers with protease inhibitors

  • Include methylation inhibitors (e.g., MS023) if studying PRMT1 activity

  • Load 20-50 μg of total protein per lane

• Gel electrophoresis:

  • 10-12% SDS-PAGE is suitable for resolving PRMT1 (40-42 kDa)

  • Include positive controls (e.g., A549 or HeLa cell lysates)

• Transfer and blocking:

  • PVDF membranes are recommended for methylated protein detection

  • Block with 5% non-fat milk or BSA in TBST

• Antibody incubation:

  • Primary: Use recommended dilutions (e.g., 1:1000-1:16000)

  • Consider overnight incubation at 4°C for optimal signal

  • Secondary: HRP-conjugated anti-rabbit IgG (typically 1:5000-1:10000)

• Detection:

  • PRMT1 typically appears as a single band at 40-42 kDa

  • Enhanced chemiluminescence (ECL) is sufficient for detection in most samples

Key considerations for troubleshooting:

• Multiple bands may indicate splice variants, degradation, or non-specific binding

• Absence of signal in positive controls suggests antibody inactivity or detection issues

• High background may require more stringent washing or antibody dilution

How should I optimize immunohistochemistry (IHC) protocols for PRMT1 detection in different tissue types?

Optimizing IHC for PRMT1 requires consideration of tissue-specific factors:

• Fixation and antigen retrieval:

  • Recommended: TE buffer pH 9.0 for optimal epitope exposure

  • Alternative: Citrate buffer pH 6.0

  • Paraffin-embedded tissues generally require heat-induced epitope retrieval

• Antibody considerations:

  • Dilution: Start with manufacturer's recommendation (1:50-1:500)

  • Incubation: Typically overnight at 4°C for best signal-to-noise ratio

  • Detection systems: Polymer-based detection systems often yield cleaner results than avidin-biotin methods

• Tissue-specific optimization:

  • Testicular tissue: PRMT1 is predominantly nuclear in spermatogonia

  • Lymphoid tissue: Higher expression in germinal centers than follicular B cells

  • Breast tissue: Both nuclear and cytosolic staining, with potential membrane localization in ER-negative tumors

  • Colon tissue: Validated for human colon tissue with TE buffer pH 9.0

• Controls:

  • Positive control: Human colon tissue shows reliable PRMT1 expression

  • Negative control: Primary antibody omission and IgG controls

  • Comparative analysis: "IHC analysis confirmed that PRMT1 is highly expressed in all BC subtypes compared to normal tissues"

• Signal interpretation:

  • PRMT1 can exhibit both nuclear and cytoplasmic localization

  • In breast cancer: "PRMT1 shows both nuclear and cytosolic staining and was also detected at the plasma membrane, mainly in ER-negative tumors"

  • In testis: "Prmt1 protein is predominantly expressed in the spermatogonial population, with markedly declined levels detectable at later stages of germ cells"

How do I interpret localization differences of PRMT1 across different cell types or experimental conditions?

PRMT1 localization varies by cell type, differentiation state, and experimental conditions:

• Normal localization patterns:

  • Primarily nuclear in spermatogonia

  • Both nuclear and cytoplasmic in many cell types

  • Predominantly cytoplasmic in some contexts

  • Potential plasma membrane association in certain cells (e.g., ER-negative breast cancer cells)

• Factors affecting localization:

  • Isoform expression: "Several PRMT1 splice variants have been described which show cytoplasmic and/or nuclear localization"

  • Cell cycle stage: Changes in localization during mitosis

  • Cellular activation: In B cells, activation leads to increased nuclear PRMT1

  • Disease state: Cancer cells may show altered PRMT1 distribution patterns

• Methodological considerations:

  • Fixation artifacts can affect apparent localization

  • Antibody epitope accessibility may differ between compartments

  • Permeabilization methods influence detection of membrane-associated PRMT1

• Interpretation guidance:

  • "PRMT1 is a well-described regulator of transcription, by methylating histones and transcription factors"

  • "PRMT1 interacts with some transmembrane receptors such as EGFR or IGF-1R"

  • "PRMT1 is expressed in both the cytosol and the nucleus in renal and pancreatic carcinomas"

When facing unexpected localization patterns, consider:

• Whether different fixation/permeabilization methods yield consistent results

• Using multiple antibodies targeting different PRMT1 epitopes

• Correlating localization with functional readouts (e.g., methylation activity)

What are common pitfalls in PRMT1 antibody experiments and how can I troubleshoot them?

Common pitfalls and their solutions include:

• Non-specific binding and high background:

  • Cause: Insufficient blocking, excessive antibody concentration

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional washing steps

  • Validation approach: "We first validated this antibody for IHC staining in a TNBC cell line (MDA-MB-468) fixed in the same method as the tissue samples"

• Lack of signal despite known PRMT1 expression:

  • Cause: Inadequate antigen retrieval, epitope masking, protein degradation

  • Solution: Optimize epitope retrieval, try alternative antibodies, include protease inhibitors

  • Sample handling: "For resting and activated, control and Prmt1^f/f CD23Cre B cells were separated by gel electrophoresis and probed for the presence of proteins containing asymmetric dimethylated arginines"

• Cross-reactivity with other PRMT family members:

  • Cause: Sequence homology, especially between PRMT1 and PRMT8 (80% homology)

  • Solution: Validate with PRMT1-knockout controls, use multiple antibodies

  • Note: "We cannot exclude that the PRMT1 antibody we used recognizes the plasma membrane-associated PRMT8, although it is brain-specific"

• Inconsistent results across experiments:

  • Cause: Batch-to-batch antibody variation, inconsistent experimental conditions

  • Solution: Use the same antibody lot when possible, standardize protocols

  • Control approach: "The type I PRMT inhibitor MS023 greatly reduced the number of aDMA-modified proteins, yielding a pattern similar to B cells from Prmt1 CD21-cre mice"

• Difficulty detecting specific PRMT1-methylated substrates:

  • Cause: Low abundance of methylated form, antibody specificity issues

  • Solution: Enrich methylated proteins via IP, use specific anti-methylarginine antibodies

  • Example approach: "Probing lysates for asymmetrically arginine-methylated proteins revealed few bands in naive B cells, but these increased in number and amount in both the GC B cell and ASC samples"

How can I assess PRMT1 enzymatic activity rather than just protein expression?

Assessing PRMT1 enzymatic activity requires specialized approaches:

• Detection of methylated substrates:

  • Anti-methylarginine antibodies: "Total cell lysates from resting and activated, control and Prmt1^f/f CD23Cre B cells were separated by gel electrophoresis and probed for the presence of proteins containing asymmetric dimethylated arginines using a specific antibody"

  • Specific methylated substrate antibodies: For example, anti-meR206-PGK1 can detect PRMT1-mediated PGK1 methylation in colorectal cancer

  • Histone methylation: "PRMT1 deficiency repressed the expression of DNA methyltransferase 1 (DNMT1) by attenuating modification of H4R3me2a and H3K27ac at enhancer regions"

• Enzymatic assays:

  • In vitro methylation assays: Using recombinant PRMT1, methyl donor (S-adenosylmethionine), and substrate proteins

  • Scintillation proximity assay: Measuring transfer of radioactive methyl groups

  • MS-based detection: Mass spectrometry to identify and quantify methylated residues

• Functional readouts:

  • Reporter assays: "PRMT1 enzymatic activity is also required to stimulate the canonical Wnt pathway"

  • Pathway activation: "PRMT1-mediated arginine asymmetric dimethylation modification of phosphoglycerate kinase 1 (PGK1) at R206 (meR206-PGK1) enhances the phosphorylation level of PGK1 at S203 (pS203-PGK1), which inhibits mitochondrial function and promotes glycolysis"

• Inhibitor-based approaches:

  • Type I PRMT inhibitors: MS023, GSK3368715, DCPT1061 can be used as tool compounds

  • Dose-response relationships: "The type I PRMT inhibitor MS023 greatly reduced the number of aDMA-modified proteins"

How can I design experiments to study PRMT1-specific methylation targets versus those shared with other PRMTs?

Distinguishing PRMT1-specific targets from those methylated by other PRMTs requires sophisticated experimental design:

• Genetic approaches:

  • PRMT1 knockout/knockdown models: "Prmt1 ablation. To test this hypothesis, we performed the qPCR analyses for PRMT family members using the testis and spleen tissues derived from WT and Prmt1-uKO mice"

  • Rescue experiments: Wild-type vs. catalytically dead PRMT1 complementation

  • Specific mutations in substrate proteins: Site-directed mutagenesis of putative methylation sites (e.g., R→K substitutions)

• Chemical approaches:

  • Selective inhibitors: "Type I PRMT inhibitors decrease breast cancer cell proliferation and show anti-tumor activity in a TNBC xenograft model"

  • Inhibitor specificity profiles: Compare effects of pan-PRMT vs. selective inhibitors

  • Dose-response relationships: Titrate inhibitor concentrations to distinguish high-affinity from low-affinity targets

• Proteomic strategies:

  • Quantitative proteomics: "We performed the immunoblotting on the Prmt1-sKO testicular lysates collected at P8 (dominated by spermatogonia) and P17 (spermatocytes) with three pan-methylarginine antibodies—MMA, ADMA and SDMA"

  • SILAC or TMT labeling: Compare methylomes between control and PRMT1-deficient samples

  • Methylation site mapping: Identify specific arginine residues methylated in substrate proteins

• Compensation mechanisms:

  • Monitor other PRMTs: "While the mRNA levels of Prmt1 were significantly down-regulated as expected, the levels for asymmetric arginine methyltransferase Prmt2/6/7, as well as the symmetric arginine methyltransferase Prmt5, were all markedly up-regulated"

  • Target overlap analysis: "Loss of Prmt1 activity spurred an eminent increase of global levels for both MMA and SDMA by other PRMT members"

Key experimental considerations:

• "Surprisingly, we discovered that the ADMA levels were significantly up-regulated as well, especially in P8 testes"

• "Consistent with prior studies in somatic cells, we found a dramatic increase in MMA levels and a moderate enhancement of SDMA methylation"

• "PRMT1 displays the highest expression levels among the PRMTs (Prmt1∼9) based on the single-cell RNA-seq analyses"

What approaches can be used to study the relationship between PRMT1 and its role in modulating signaling pathways in cancer?

To investigate PRMT1's role in cancer signaling pathways:

• Transcriptomic and ChIP analyses:

  • Gene expression profiling: "Transcriptomic analysis and chromatin immunoprecipitation revealed that PRMT1 regulates the epidermal growth factor receptor (EGFR) and the Wnt signaling pathways, reported to be activated in TNBC"

  • ChIP-seq: "PRMT1 was directly recruited to two promoter regions of EGFR in MDA-MB-468 cells"

  • Pathway enrichment analysis: "Our microarray analysis revealed two key players of the Wnt signaling pathway, LRP5 and PORCN (Porcupine), to be less expressed following PRMT1 depletion"

• Functional genomics approaches:

  • CRISPR/Cas9 screening: Identify synthetic lethal interactions with PRMT1

  • shRNA/siRNA: "PRMT1 depletion (i) decreased the cell viability, (ii) blocked their clonogenic potential, and (iii) induced DNA damage and apoptosis in various cell lines of different BC subtypes"

  • Methylation-deficient mutants: Introduce substrate proteins with R→K mutations at PRMT1 target sites

• Pathway-specific readouts:

  • EGFR signaling: "PRMT1 depletion also decreased EGFR protein expression"

  • Wnt signaling: "PRMT1 enzymatic activity is also required to stimulate the canonical Wnt pathway"

  • Glycolysis: "PRMT1-mediated arginine asymmetric dimethylation modification of phosphoglycerate kinase 1 (PGK1) at R206 (meR206-PGK1) enhances the phosphorylation level of PGK1 at S203 (pS203-PGK1), which inhibits mitochondrial function and promotes glycolysis"

  • Interferon response: "PRMT1 deficiency or inhibition with DCPT1061 significantly restrained refractory melanoma growth and increased intratumoral CD8+ T cells"

• Combination therapy studies:

  • Synergy testing: "These inhibitors display synergistic interactions with some chemotherapies used to treat TNBC patients as well as erlotinib, an EGFR inhibitor"

  • In vivo models: "PRMT1 inhibition with DCPT1061 synergized with PD-1 blockade to suppress tumor progression and increase the proportion of CD8+ T cells as well as IFNγ+CD8+ T cells in vivo"

  • Clinical correlation: "meR206-PGK1 expression is positively correlated with the poor survival of patients with colorectal cancer"