PRMT2 Antibody

What is PRMT2 and why is it important to study?

PRMT2, also known as HRMT1L1, is a member of the arginine methyltransferase family that catalyzes arginine methylation and regulates diverse cellular processes including transcription, translation, DNA repair, and RNA processing. It is responsible for the H3R8me2a histone mark, which is associated with active transcription . PRMT2 interacts with many key regulatory proteins including Rb, NF-κB, ER-alpha, and androgen receptor, positioning it as a critical player in various cellular pathways . Recent research has implicated PRMT2 in several disease processes, including its overexpression in cancers such as hepatocellular carcinoma, glioblastoma, and renal cell carcinoma . Additionally, PRMT2 has been identified as a host restriction factor for HIV-1 transcription, promoting viral latency through methylation of the viral Tat protein .

What applications are PRMT2 antibodies commonly used for?

PRMT2 antibodies are versatile tools employed across multiple experimental platforms:

These applications enable researchers to detect PRMT2 expression, localization, and interactions in various experimental systems, including cell lines, tissue samples, and protein extracts .

What are the differences between monoclonal and polyclonal PRMT2 antibodies?

The choice between monoclonal and polyclonal PRMT2 antibodies depends on your specific research requirements:

Monoclonal antibodies (e.g., 66885-1-Ig) offer:

• Superior lot-to-lot consistency

• Higher specificity for a single epitope

• Reduced background in applications like immunofluorescence

• Ideal for quantitative assays and detecting specific epitopes

Polyclonal antibodies offer:

• Recognition of multiple epitopes on PRMT2

• Often higher sensitivity in applications like Western blotting

• More robust detection across different species

• Better tolerance of protein denaturation or modifications

What is the expected molecular weight for PRMT2 in Western blotting?

PRMT2 typically appears at 45-50 kDa in Western blot applications, which is slightly lower than its calculated molecular weight of 49 kDa . This slight discrepancy between calculated and observed molecular weights is common for many proteins due to post-translational modifications, protein folding, or the specific gel system used. Some splice variants of PRMT2 have been identified in breast cancers, which may appear at different molecular weights . When validating a new PRMT2 antibody, researchers should compare their observed band against both positive controls (e.g., SKOV-3 cells, HeLa cells, Jurkat cells) and negative controls (e.g., PRMT2 knockout or knockdown samples) .

Which cell lines or tissues serve as positive controls for PRMT2 detection?

Multiple validated positive controls have been documented for PRMT2 antibody applications:

For Western blotting:

• SKOV-3 cells

• HeLa cells

• Jurkat cells

• LNCaP cells

• 4T1 cells

• Rat heart tissue

For immunohistochemistry:

• Human thyroid cancer tissue (with recommended antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0)

For immunofluorescence:

• HeLa cells

When establishing a new experimental system, these validated sources provide reliable positive controls for antibody optimization.

How can I validate the specificity of a PRMT2 antibody?

Rigorous validation ensures reliable PRMT2 detection across applications:

• Knockdown/knockout validation: Compare detection between wild-type samples and those with PRMT2 knockdown using siRNA (e.g., PRMT2-specific siRNA-A from Invitrogen or siRNA-B from Sigma) or CRISPR/Cas9 knockout . Complete signal loss in knockout samples confirms specificity.

• Overexpression controls: Compare detection in cells with endogenous PRMT2 versus those overexpressing PRMT2 to confirm proper molecular weight and signal increase .

• Cross-reactivity assessment: Test the antibody against samples from multiple species if cross-reactivity is claimed. Many PRMT2 antibodies show reactivity with human, mouse, and rat samples .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to confirm that the peptide blocks specific binding .

• Multiple antibody validation: Compare results using antibodies recognizing different epitopes of PRMT2 (e.g., N-terminal versus C-terminal) .

What are the optimal sample preparation methods for PRMT2 detection?

Effective sample preparation is crucial for successful PRMT2 detection:

For Western blotting:

• Use RIPA buffer or NP-40 buffer with protease inhibitors

• Include phosphatase inhibitors if studying PRMT2 phosphorylation

• Add deacetylase inhibitors if studying acetylation status

• Include 1-5% SDS for membrane protein solubilization

• Maintain samples at 4°C during lysis to prevent degradation

For immunofluorescence:

• 4% paraformaldehyde fixation (10-15 minutes at room temperature)

• Permeabilization with 0.1-0.5% Triton X-100

• Blocking with 5% normal serum corresponding to secondary antibody species

For immunohistochemistry:

• Antigen retrieval with TE buffer pH 9.0 is recommended

• Alternative: citrate buffer pH 6.0

• 4-5 μm FFPE tissue sections typically yield optimal results

What are the storage and handling recommendations for PRMT2 antibodies?

Proper handling ensures maximum antibody performance and longevity:

For optimal performance, allow the antibody to equilibrate to room temperature before opening the vial, and briefly centrifuge before use to collect the solution at the bottom of the tube .

How can PRMT2 antibodies be utilized to study its role in HIV-1 latency?

Recent research has identified PRMT2 as a host restriction factor for HIV-1 transcription and proviral reactivation . Researchers studying this mechanism can employ PRMT2 antibodies in several sophisticated approaches:

• Detecting PRMT2-Tat interactions: Co-immunoprecipitation using PRMT2 antibodies can pull down HIV-1 Tat protein to study their physical association. Studies have shown that Tat is physically associated with PRMT2 and preferentially methylated at the R52 residue .

• Mapping nucleolar localization: Immunofluorescence co-staining with PRMT2 antibodies and nucleolar markers like NPM1 can visualize how PRMT2 enhances Tat association with NPM1, causing its nucleolar sequestration .

• Analyzing methylation status: Using custom antibodies that specifically recognize R52ame2 Tat (such as TatR52ame2_2) alongside PRMT2 antibodies helps track the methylation status of Tat in latently infected cells .

• Monitoring latency establishment: In dual-color HIV-1 reporter systems, correlating PRMT2 levels with GFP/mKO2 expression can demonstrate how PRMT2 promotes latency establishment during viral infection .

• Evaluating latency reversal agents (LRAs): PRMT2 antibodies can assess how PRMT2 inhibition might synergize with existing LRAs to reactivate latent provirus in patient-derived CD4+ T cells .

This approach has revealed that the methylase activity of PRMT2 is critical for inhibiting Tat transactivation and maintaining proviral latency, suggesting potential for PRMT2 inhibitors in HIV cure strategies .

What role does PRMT2 play in cancer progression and how can antibodies help study these mechanisms?

PRMT2 has been implicated in several cancer types, with antibody-based studies revealing key mechanisms:

In renal cell carcinoma (RCC):

• PRMT2 is upregulated in primary RCC and RCC cell lines

• Overexpression promotes RCC cell proliferation and motility both in vitro and in vivo

• PRMT2 mediates H3R8 asymmetric dimethylation (H3R8me2a) in the WNT5A promoter region

• This enhances WNT5A transcriptional expression, activating Wnt signaling pathway

In other cancer types:

• PRMT2 is overexpressed in hepatocellular carcinoma and glioblastoma

• Its catalytic activity increases tumorigenesis

• Splice variants of PRMT2 have been described in breast cancers

Antibody-based methodologies to study these mechanisms include:

• Chromatin immunoprecipitation (ChIP) using PRMT2 antibodies to identify genomic targets

• Immunohistochemistry to correlate PRMT2 expression with clinical outcomes

• Co-immunoprecipitation to study interaction partners in cancer contexts

• Immunoblotting of tumor samples to quantify expression across cancer stages

These approaches have revealed PRMT2 as a potential therapeutic target in multiple cancer types through its epigenetic regulatory functions.

How can I detect PRMT2-mediated histone modifications using antibodies?

PRMT2 catalyzes the H3R8me2a (histone H3 asymmetric dimethylarginine 8) mark, which is associated with active transcription . Detecting this specific modification requires a combination of PRMT2 antibodies and modification-specific antibodies:

• ChIP-seq approach:

  • Perform chromatin immunoprecipitation using anti-H3R8me2a antibodies

  • In parallel, perform ChIP with PRMT2-specific antibodies

  • Compare binding profiles to identify regions where both PRMT2 and H3R8me2a co-localize

  • Validate with qPCR at specific loci of interest (e.g., WNT5A promoter in RCC cells)

• Sequential ChIP (re-ChIP):

  • First immunoprecipitate with PRMT2 antibodies

  • Perform a second immunoprecipitation on the eluted material using H3R8me2a antibodies

  • This identifies genomic regions where PRMT2 directly mediates H3R8me2a modification

• Immunofluorescence co-localization:

  • Co-stain cells with PRMT2 antibodies and H3R8me2a antibodies

  • Analyze co-localization patterns using confocal microscopy

  • Quantify co-localization coefficients across different cellular conditions

When conducting these experiments, include appropriate controls such as PRMT2 knockdown/knockout samples to confirm the specificity of the detected modifications .

What approaches can differentiate between PRMT2 splice variants using antibodies?

PRMT2 splice variants have been described particularly in breast cancers , and differentiating between these variants requires careful antibody selection and experimental design:

• Epitope-specific antibodies:

  • Select antibodies targeting regions that are differentially present in splice variants

  • For N-terminal variants, use antibodies targeting amino acids 1-300 or 22-53

  • For C-terminal variants, use antibodies targeting amino acids 344-375

  • Compare detection patterns across multiple antibodies recognizing different regions

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate PRMT2 using antibodies that recognize all variants

  • Analyze the precipitated proteins by mass spectrometry to identify peptides unique to specific variants

  • Quantify the relative abundance of variant-specific peptides

• RT-PCR validation:

  • Complement antibody-based detection with RT-PCR using variant-specific primers

  • Correlate protein detection with mRNA expression profiles

  • This dual approach provides stronger evidence for variant expression

• Size-based discrimination:

  • Use high-resolution SDS-PAGE to separate variants by molecular weight

  • Different variants may show distinct migration patterns due to size differences

  • Follow with Western blotting using pan-PRMT2 antibodies to detect all variants simultaneously

These approaches can help researchers understand the differential expression and functions of PRMT2 splice variants in normal and disease states.

How can I optimize PRMT2 antibodies for chromatin immunoprecipitation (ChIP) applications?

Optimizing PRMT2 antibodies for ChIP requires careful consideration of several factors:

• Antibody selection:

  • Choose antibodies validated for ChIP applications, such as those listed in search result

  • Monoclonal antibodies often provide higher specificity and reproducibility

  • Confirm the antibody recognizes native (non-denatured) PRMT2

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-1.5%) and crosslinking times (5-15 minutes)

  • For indirect interactions, consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Sonication parameters:

  • Optimize sonication conditions to yield chromatin fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis before proceeding

• Antibody concentration titration:

  • Perform preliminary ChIP with different antibody amounts (2-10 μg per reaction)

  • Analyze enrichment by qPCR at known PRMT2 binding sites

  • The optimal concentration should maximize signal-to-noise ratio

• Positive control loci:

  • Include primers for regions known to be bound by PRMT2, such as the WNT5A promoter in RCC cells

  • Compare enrichment at these sites to negative control regions

• Pre-clearing and blocking:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Include sufficient blocking agents (BSA, salmon sperm DNA) to minimize non-specific binding

• Washing stringency:

  • Test different washing buffer stringencies to optimize signal-to-noise ratio

  • More stringent washes reduce background but may reduce signal from weak interactions

This optimized protocol will enable reliable mapping of PRMT2 genomic binding sites and correlation with its methyltransferase activity.

What technical challenges exist in using PRMT2 antibodies for co-immunoprecipitation of interaction partners?

Co-immunoprecipitation (co-IP) of PRMT2 and its binding partners presents several technical challenges:

• Preserving native interactions:

  • PRMT2 interactions with partners like PRMT1, Rb, NF-κB, and steroid receptors may be sensitive to extraction conditions

  • Use gentle lysis buffers (e.g., NP-40 or digitonin-based) that maintain protein-protein interactions

  • Avoid harsh detergents like SDS that can disrupt protein complexes

• Nuclear protein extraction:

  • PRMT2 shuttles between cytoplasm and nucleus, requiring effective nuclear extraction

  • Use nuclear extraction kits or protocols that maintain protein complexes

  • Verify subcellular fractionation efficiency by immunoblotting for compartment-specific markers

• Antibody orientation:

  • Direct IP using PRMT2 antibodies vs. reverse IP using antibodies against interaction partners may yield different results

  • Compare both approaches to obtain comprehensive interaction data

  • The choice of antibody (e.g., 67007 for IP at 1:50 dilution) can significantly impact results

• Distinguishing direct vs. indirect interactions:

  • Include RNase/DNase treatment to eliminate nucleic acid-mediated associations

  • Consider using crosslinking approaches to stabilize transient interactions

  • Validate direct interactions using purified recombinant proteins

• Detecting methylated partners:

  • When studying PRMT2's methyltransferase activity on partners, include methyl-specific antibodies in the analysis

  • For example, when studying Tat methylation, specific antibodies like TatR52ame2_2 can be used

• Verifying specificity:

  • Include appropriate negative controls (IgG, PRMT2 knockout/knockdown)

  • Confirm interactions using multiple antibodies targeting different epitopes of PRMT2

  • Validate key interactions with orthogonal methods (proximity ligation assay, FRET)

By addressing these challenges, researchers can reliably identify and characterize the interactome of PRMT2 in various biological contexts.

How can PRMT2 antibodies be used to evaluate the efficacy of PRMT2 inhibitors in research?

As potential PRMT2 inhibitors are developed, antibody-based approaches will be crucial for evaluating their efficacy:

• Target engagement assays:

  • Cellular thermal shift assay (CETSA) using PRMT2 antibodies can confirm inhibitor binding to PRMT2 in cells

  • Drug affinity responsive target stability (DARTS) can assess protective effects of inhibitors against protease digestion

• Functional inhibition assessment:

  • Immunoblotting for H3R8me2a levels can evaluate inhibition of PRMT2's methyltransferase activity

  • In HIV-1 models, measure R52 methylation of Tat using specific antibodies like TatR52ame2_2

  • These direct readouts of enzymatic activity provide reliable measures of inhibitor efficacy

• Pathway modulation:

  • In RCC models, monitor WNT5A expression and Wnt pathway activation as downstream effects of PRMT2 inhibition

  • In HIV-1 models, assess nucleolar vs. nucleoplasmic distribution of Tat using immunofluorescence

• Phenotypic reversal:

  • Compare inhibitor effects with PRMT2 knockdown/knockout phenotypes to confirm on-target activity

  • In cancer models, assess proliferation and motility inhibition

  • In HIV-1 latency models, measure proviral reactivation

• Resistance mechanism identification:

  • Immunoprecipitation followed by mass spectrometry can identify changes in PRMT2 interaction partners upon inhibitor treatment

  • These analyses may reveal compensatory mechanisms or resistance pathways

Although selective PRMT2 inhibitors are not yet commercially available, the structural information from zebrafish and mouse PRMT2 should facilitate inhibitor development, with antibody-based approaches being essential for their evaluation .

What methodological approaches can overcome antibody cross-reactivity with other PRMT family members?

The PRMT family contains nine members with similar catalytic domains, creating potential cross-reactivity challenges:

• Epitope selection:

  • Choose antibodies targeting unique regions of PRMT2, such as its N-terminal SH3 domain which is not present in other PRMTs

  • Antibodies recognizing amino acids 22-53 or 344-375 may offer higher specificity than those targeting the conserved catalytic domain

• Validation in knockout systems:

  • Test antibodies in PRMT2 knockout systems to ensure complete loss of signal

  • Also test in cells overexpressing other PRMT family members to check for cross-reactivity

  • CRISPR/Cas9 knockout of PRMT2 provides an ideal negative control

• Immunodepletion strategy:

  • Sequentially deplete lysates with antibodies against other PRMT family members before PRMT2 detection

  • This approach can help isolate PRMT2-specific signals from potential cross-reactive signals

• Specific methylation patterns:

  • Focus on PRMT2-specific methylation targets (H3R8me2a) rather than shared targets

  • Compare methylation patterns with those of other PRMTs (e.g., PRMT1-mediated H4R3me2a)

  • This functional readout can complement direct PRMT2 detection

• Isoform-specific assays:

  • Design assays that detect PRMT2-specific products or activities

  • For example, monitoring WNT5A expression in RCC models provides a PRMT2-specific functional readout

These methodological approaches allow researchers to distinguish PRMT2-specific signals from those of other PRMT family members, ensuring reliable data interpretation in complex biological systems.