PRMT11 Antibody

What is PRMT11 and how does it relate to PRMT1?

PRMT11 (AtPRMT11) is an Arabidopsis arginine methyltransferase that was discovered as an interaction partner of AtMBD7, a methyl-DNA-binding protein containing three MBD domains. AtPRMT11 is highly similar to mammalian PRMT1 and functions as a type I arginine methyltransferase that creates asymmetrically dimethylated arginines. The relationship between PRMT11 and AtMBD7 suggests an important link between DNA methylation and arginine methylation in plants . In mammalian systems, PRMT1 is the predominant type I arginine methyltransferase and is more commonly studied than plant PRMT11, though they share functional similarities in their respective organisms.

What are the major functions of PRMT1/PRMT11 in cellular processes?

In plants, AtPRMT11 acts as an arginine methyltransferase active on both histones and proteins in cellular extracts. It post-translationally modifies AtMBD7 protein, specifically at the C-terminal methylated DNA-binding domain . In mammals, PRMT1 plays crucial roles in multiple cellular processes:

• B cell development: PRMT1 regulates B cell fate after activation, promoting antibody affinity maturation by favoring dark zone fate and proliferation while limiting differentiation

• Gene expression regulation: Through methylation of histones and transcription factors

• Cell cycle control: Influencing proliferation in both normal and cancer cells

• Cancer progression: PRMT1 expression in B cell lymphoma correlates with poor disease outcomes and depends on MYC and mTORC1 activity

How is PRMT1 expression regulated in different cell types?

PRMT1 expression varies across different cell types and developmental stages. In B cells, Prmt1 mRNA levels are high during early B cell development and peak again in activated and germinal center B cells (GCBC). Within the light zone (LZ) of germinal centers, Prmt1 is substantially upregulated in GCBC subsets with high Myc expression . This regulation appears to involve the MYC transcription factor and mTORC1 signaling pathways, which are critical regulators of cell growth and metabolism. The dynamic expression pattern suggests tight control of PRMT1 levels corresponding to specific cellular functions during B cell development and immune responses.

What are the validated applications for PRMT1 antibodies in research?

Based on validated research applications, PRMT1 antibodies can be used in multiple experimental contexts:

How can PRMT1 antibodies be used to identify methylated target proteins?

PRMT1 antibodies can be used in combination with antibodies against asymmetrically dimethylated arginine (ASYM24) in a multi-step approach to identify methylated target proteins:

• Two-dimensional gel electrophoresis (2D-GE): Separate proteins from cell or tissue samples based on isoelectric point and molecular weight.

• Two-dimensional Western blotting (2D-WB): Transfer separated proteins to membranes and probe with anti-ASYM24 antibody to detect proteins containing asymmetrically dimethylated arginines.

• Match protein spots: Compare the 2D-WB pattern with a Coomassie blue-stained gel of the same sample.

• Mass spectrometry analysis: Excise matching spots from the gel and identify proteins using mass spectrometry.

• Validation: Confirm methylation status using targeted pulldown experiments with PRMT1 antibodies .

This approach has successfully identified several PRMT substrates in colorectal cancer cells, including CACYBP, GLOD4, MAPRE1, CCT7, TKT, CK8, and HSPA8 .

What are the recommended dilutions for different experimental applications of PRMT1 antibodies?

The optimal dilutions for PRMT1 antibody applications vary by technique:

It is recommended to titrate the antibody in each testing system to obtain optimal results, as sample-dependent variations may occur.

How should researchers distinguish between PRMT11 (plant) and PRMT1 (mammalian) in their experimental designs?

When designing experiments involving PRMTs, researchers should consider these key distinctions:

• Organism specificity: PRMT11 is specific to Arabidopsis and other plants, while PRMT1 is found in mammals. Ensure your antibody is specific to the appropriate species.

• Sequence homology assessment: Prior to experiments, perform sequence alignment to evaluate homology between your target PRMT and the epitope recognized by your antibody.

• Validation controls:

  • For plant studies: Use Arabidopsis T-DNA mutant lines lacking AtPRMT11 mRNA as negative controls

  • For mammalian studies: Use PRMT1 knockout or knockdown cells (e.g., using CRISPR-Cas9 or siRNA)

• Cross-reactivity testing: If working with novel species or variants, validate antibody specificity using recombinant proteins or overexpression systems.

• Literature verification: Consult species-specific literature to ensure experimental designs align with known biology of the target PRMT in your model system.

What are the critical factors for successful immunoprecipitation with PRMT1 antibodies?

Successful immunoprecipitation of PRMT1 requires careful attention to several factors:

• Lysis buffer composition: Use buffers that preserve protein-protein interactions while effectively extracting nuclear proteins:

  • RIPA buffer with 150-300 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, and protease inhibitors

  • Consider including phosphatase inhibitors if studying phosphorylation-dependent interactions

• Antibody selection: Choose antibodies validated for IP applications (as indicated in result )

• Protein amount optimization: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-clearing: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding

• Incubation conditions: Optimize antibody-protein binding by incubating at 4°C overnight with gentle rotation

• Washing stringency: Balance between removing non-specific interactions and preserving true interactions

• Elution methods: Consider native elution with competing peptides for downstream functional assays, or denaturing elution for identification purposes

How can researchers detect and quantify arginine methylation activity in their experimental systems?

Researchers can employ several methodologies to detect and quantify arginine methylation:

• In vitro methyltransferase assays:

  • Use recombinant PRMT1/PRMT11 with potential substrates and S-adenosyl-L-[methyl-³H]methionine

  • Measure incorporation of radioactive methyl groups via liquid scintillation counting

  • Alternative: Use non-radioactive S-adenosyl-L-methionine and detect methylation with ASYM24 antibodies

• Antibody-based detection in cells/tissues:

  • Western blot with ASYM24 antibodies to detect asymmetrically dimethylated arginines

  • Compare patterns between wild-type and PRMT-deficient samples to identify specific substrates

• Mass spectrometry approaches:

  • Enrich methylated peptides using anti-methyl-arginine antibodies

  • Perform liquid chromatography-mass spectrometry (LC-MS/MS) to identify methylated residues

  • Use SILAC or TMT labeling for quantitative comparison between conditions

• Genetic approaches:

  • Analyze methylation patterns in T-DNA mutant lines lacking AtPRMT11 mRNA (for plants)

  • Use conditional knockout models to study tissue-specific effects of PRMT deficiency

How does PRMT1 regulate the balance between proliferation and differentiation in B cells?

PRMT1 plays a sophisticated role in regulating B cell fate decisions after activation:

• Promotion of dark zone (DZ) fate: PRMT1 expression favors DZ fate over light zone (LZ) fate in germinal center B cells, promoting continued affinity maturation rather than differentiation. This is achieved through:

  • Regulation of cell cycle genes to maintain proliferative capacity

  • Supporting somatic hypermutation machinery in DZ cells

  • Facilitating LZ to DZ recycling for continued affinity maturation

• Restriction of plasma cell differentiation: PRMT1 intrinsically limits plasma cell differentiation, which appears to be a function co-opted by B cell lymphoma cells to maintain their undifferentiated state. This suggests PRMT1 may regulate key transcription factors that control B cell differentiation .

• Memory B cell generation: PRMT1 deficiency results in enhanced memory B cell generation, though these cells show quality defects due to compromised germinal center reactions .

• Molecular mechanisms: Evidence suggests PRMT1 may function through both:

  • Histone modifications that influence chromatin accessibility

  • Direct methylation of non-histone proteins involved in B cell fate decisions

Understanding these mechanisms has significant implications for both normal immune function and B cell malignancies.

What is the relationship between DNA methylation and arginine methylation suggested by PRMT11-MBD7 interactions?

The interaction between AtPRMT11 and AtMBD7 in Arabidopsis suggests a fascinating potential link between DNA methylation and arginine methylation as epigenetic regulatory mechanisms:

• MBD7 as a methyl-DNA binding protein: AtMBD7 is unique in containing three methyl-DNA-binding domains, allowing it to recognize and bind methylated DNA sequences .

• PRMT11 as a modification enzyme: AtPRMT11 post-translationally modifies AtMBD7 at its C-terminal methylated DNA-binding domain through arginine methylation .

• Potential regulatory circuit: This interaction suggests a regulatory circuit where:

  • MBD7 binds to methylated DNA regions

  • PRMT11 modifies MBD7 through arginine methylation

  • Modified MBD7 may exhibit altered binding properties or recruit different protein complexes

  • This could influence gene expression at methylated DNA loci

• Evolutionary implications: This link between two major epigenetic mechanisms (DNA methylation and protein arginine methylation) appears to be conserved from plants to mammals, suggesting fundamental importance in chromatin regulation.

• Research gaps: Further studies are needed to determine:

  • How arginine methylation affects MBD7 binding affinity to methylated DNA

  • Whether this interaction is regulated by developmental or environmental signals

  • If similar mechanisms exist in mammalian cells between PRMT1 and MBD proteins

How can researchers distinguish between different PRMT-mediated methylation patterns in their experimental systems?

Distinguishing between different PRMT-mediated methylation patterns requires sophisticated approaches:

• Antibody selection for different methylation types:

  • Type I PRMTs (including PRMT1/PRMT11): Use antibodies specific for asymmetric dimethylarginine (ADMA), such as ASYM24

  • Type II PRMTs: Use antibodies specific for symmetric dimethylarginine (SDMA)

  • Type III PRMTs: Use antibodies specific for monomethylarginine (MMA)

• Mass spectrometry approaches:

  • Characteristic mass shifts: ADMA (+28.0313 Da), SDMA (+28.0313 Da), MMA (+14.0157 Da)

  • Fragmentation patterns: ADMA and SDMA produce distinctive MS/MS fragmentation patterns

  • Specialized methods like electron transfer dissociation (ETD) can improve methylarginine site localization

• Chemical approaches:

  • Selective chemical derivatization methods can distinguish between methylation types

  • Hydrolysis followed by HPLC can separate and quantify different methylarginine species

• Genetic tools:

  • Use cells deficient in specific PRMTs (e.g., PRMT1 knockout for type I methylation)

  • Employ specific PRMT inhibitors: Type I (e.g., MS023), Type II (e.g., GSK591)

  • Compare methylation patterns between wild-type and PRMT-deficient systems

• Sequential immunoprecipitation:

  • First IP with a general methyl-arginine antibody

  • Second IP with type-specific antibodies to enrich for specific methylation patterns

  • Analyze the enriched proteins to determine their methylation status

This multi-faceted approach allows researchers to precisely characterize the methylation landscape in their experimental systems.

What are common sources of non-specific binding with PRMT1 antibodies and how can they be mitigated?

Researchers frequently encounter non-specific binding issues when working with PRMT1 antibodies. These can be addressed through several approaches:

• Antibody selection and validation:

  • Choose antibodies validated with knockout/knockdown controls

  • Verify specificity using recombinant PRMT1 protein

  • Assess cross-reactivity with other PRMT family members

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extend blocking time to reduce background

  • Consider adding 0.1-0.5% Tween-20 to blocking buffer

• Sample preparation:

  • Pre-clear lysates before immunoprecipitation

  • Use freshly prepared samples to minimize protein degradation

  • Consider protein extraction methods optimized for nuclear proteins

• Washing conditions:

  • Increase washing stringency (higher salt, more detergent)

  • Extend washing time for immunoprecipitation experiments

  • Use TBS-T with optimized Tween-20 concentration for immunoblotting

• Antibody concentration:

  • Titrate antibody to determine optimal concentration

  • For Western blotting, use the recommended 1:2000-1:16000 dilution range

  • For IHC, use 1:50-1:500 with appropriate antigen retrieval

• Controls to include:

  • Isotype control antibodies

  • PRMT1 knockdown/knockout samples

  • Peptide competition assays to confirm specificity

What are the key considerations when designing experiments to study PRMT1-mediated arginine methylation in cancer contexts?

When investigating PRMT1-mediated arginine methylation in cancer research, several key considerations should guide experimental design:

• Selection of appropriate models:

  • Compare matched tumor/normal tissues from the same patient

  • Include cell lines with varying PRMT1 expression levels

  • Consider patient-derived xenografts for more clinically relevant models

• Control for expression vs. activity:

  • Measure both PRMT1 protein levels and methyltransferase activity

  • Assess global arginine methylation patterns with ASYM24 antibodies

  • Compare enzyme activity with protein expression to identify discrepancies

• Target identification approaches:

  • Implement 2D-GE combined with 2D-WB and mass spectrometry

  • Enrich methylated proteins using ASYM24 antibodies

  • Focus on cancer-relevant pathways based on methylated targets identified

• Validation of findings:

  • Confirm methylation sites by site-directed mutagenesis

  • Use PRMT1 inhibitors to establish causality

  • Perform functional assays to determine the consequences of methylation

• Clinical correlations:

  • Analyze PRMT1 expression and target methylation in patient samples

  • Correlate with clinical parameters (stage, grade, survival)

  • Assess PRMT1 expression alongside MYC and mTORC1 activity markers

• Therapeutic implications:

  • Test PRMT1 inhibitors alone and in combination with standard treatments

  • Identify patient subgroups likely to benefit from PRMT inhibition

  • Explore resistance mechanisms to PRMT inhibition

What emerging technologies might enhance the study of PRMT1/PRMT11-mediated arginine methylation?

Several cutting-edge technologies show promise for advancing PRMT research:

• Proteome-wide methods:

  • Advanced mass spectrometry approaches for comprehensive methylome analysis

  • Proximity labeling methods (BioID, APEX) to identify methyltransferase interaction networks

  • CRISPR screens to identify genes involved in PRMT1 regulation

• Single-cell approaches:

  • Single-cell proteomics to examine cell-to-cell variation in methylation patterns

  • Single-cell RNA-seq to correlate transcriptional changes with PRMT1 activity

  • Spatial transcriptomics to map PRMT1 activity in tissue contexts

• Structural biology advances:

  • Cryo-EM studies of PRMT1 complexes to understand substrate recognition

  • Hydrogen-deuterium exchange mass spectrometry to probe dynamic interactions

  • AlphaFold and related computational approaches to predict methylation sites

• Live-cell methylation monitoring:

  • Development of methylation-sensitive fluorescent reporters

  • Optogenetic control of PRMT1 activity to study temporal aspects

  • Real-time monitoring of methylation dynamics

• Therapeutic development:

  • Structure-guided design of isoform-specific PRMT inhibitors

  • Targeted protein degradation approaches (PROTACs) for selective PRMT depletion

  • Combination therapy strategies with epigenetic modulators

These emerging technologies promise to provide deeper insights into the complex roles of PRMTs in normal physiology and disease.

How might differential PRMT1 expression across cancer types inform potential therapeutic strategies?

The differential expression and activity of PRMT1 across cancer types offers important insights for therapeutic development:

• Cancer-specific expression patterns:

  • B cell lymphomas show high PRMT1 expression correlating with poor outcomes

  • Colorectal cancers exhibit elevated asymmetric arginine dimethylation

  • Expression patterns may vary by cancer subtype and stage

• Mechanistic dependencies:

  • MYC and mTORC1 activity regulate PRMT1 expression in B cell lymphomas

  • These pathways may predict sensitivity to PRMT1 inhibition

  • Combined targeting of these regulatory pathways with PRMT1 inhibition may be synergistic

• Functional consequences:

  • PRMT1 promotes proliferation while limiting differentiation in B cell contexts

  • Similar phenotypes in solid tumors would suggest broad applicability of PRMT1 inhibition

  • Cancer types with stem cell-like features may be particularly dependent on PRMT1

• Biomarker development:

  • Asymmetric dimethylarginine levels may serve as biomarkers for PRMT1 activity

  • Specific PRMT1 substrates (e.g., MAPRE1, CCT7, TKT, HSPA8 in colorectal cancer) could indicate PRMT1 dependency

  • Expression ratios between PRMT1 and its substrates might predict treatment response

• Precision medicine approaches:

  • Match PRMT1 inhibitors to cancers with high dependency on arginine methylation

  • Develop combination strategies based on cancer-specific methylation targets

  • Use methylation patterns to stratify patients for clinical trials

Understanding these cancer-specific patterns may lead to more effective and personalized therapeutic strategies targeting PRMT1 and arginine methylation.