PRMT6 Monoclonal Antibody

What is PRMT6 and why is it important in cellular research?

PRMT6 is a 41.9 kDa type I protein arginine methyltransferase predominantly localized in the nucleus, in contrast to other PRMTs that may be found in both nucleus and cytosol or predominantly in the cytoplasm . It plays critical roles in epigenetic regulation of gene expression, alternative splicing, development and differentiation, DNA repair, cell proliferation and senescence, DNA methylation, mitosis, inflammation, and innate antiviral immunity . PRMT6 is particularly significant because it generates asymmetric dimethylation modifications in histones (H3R2me2a, H3R17me2a, H3R42me2a, and H2AR26me2a), directly influencing gene expression through epigenetic mechanisms . Additionally, PRMT6 has demonstrated significant antiviral activity against HIV-1 by methylating and impairing various HIV-1 proteins .

What are the key considerations when selecting a PRMT6 monoclonal antibody?

When selecting a PRMT6 monoclonal antibody, researchers should consider:

• Experimental application compatibility (Western blot, IHC, flow cytometry, etc.)

• Species reactivity required for your samples

• Epitope location and accessibility in your experimental conditions

• Clonality (monoclonal vs. polyclonal) based on specificity requirements

• Validation data available from the manufacturer

For example, the Mouse Monoclonal PRMT6 antibody (67981-1-PBS) has been validated for Western blot and Indirect ELISA applications with reactivity against human, mouse, and rat samples . Similarly, Thermo Fisher's PRMT6 Monoclonal Antibody (PCRP-PRMT6-2C9) shows predicted reactivity with rat samples . Select antibodies with validation in your specific application to minimize experimental issues.

How do monoclonal and polyclonal PRMT6 antibodies compare in research applications?

The comparison below outlines the performance differences between monoclonal and polyclonal PRMT6 antibodies:

Choose monoclonal antibodies when absolute specificity is crucial or when performing quantitative analyses. Select polyclonal antibodies when maximum sensitivity is needed, particularly with low-abundance targets.

What are the validated applications for PRMT6 monoclonal antibodies?

PRMT6 monoclonal antibodies have been validated for several research applications:

• Western Blotting (WB): Most PRMT6 antibodies are validated for WB, with recommended dilutions typically between 1:500-1:1000 .

• Flow Cytometry: Some monoclonal antibodies like ab277102 are suitable for flow cytometry applications .

• Immunohistochemistry (IHC-P): Selected antibodies like the Rabbit Polyclonal ab72205 are validated for paraffin-embedded sections .

• Protein Arrays: Monoclonal antibodies such as ab277102 have been validated for protein array applications .

• ELISA: Certain antibodies like 67981-1-PBS are validated for indirect ELISA methods .

When selecting an antibody for your specific application, verify that the manufacturer has validated it for your intended use. For example, the Mouse Monoclonal 67981-1-PBS is validated for WB and indirect ELISA, showing reactivity with human, mouse, and rat samples .

What is the optimal protocol for Western blotting using PRMT6 monoclonal antibodies?

For optimal Western blot results with PRMT6 monoclonal antibodies:

• Sample preparation:

  • Prepare cell/tissue lysates in RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

  • For nuclear proteins like PRMT6, ensure proper nuclear extraction

• Gel electrophoresis and transfer:

  • Load 20-30μg of protein per lane

  • Use 10-12% SDS-PAGE gels (PRMT6 is approximately 42 kDa)

  • Transfer to PVDF membrane (preferred over nitrocellulose for nuclear proteins)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary PRMT6 antibody at recommended dilution (typically 1:500-1:1000)

  • For Mouse Monoclonal antibodies like 67981-1-PBS, incubate overnight at 4°C

• Detection:

  • Wash thoroughly with TBST (3-5 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Develop using ECL substrate and image

• Expected results:

  • PRMT6 should be detected at approximately 42 kDa

  • Validated in cell lines including HeLa, MCF7, NIH/3T3, and Jurkat

This protocol has been optimized based on the recommendations for PRMT6 antibodies from multiple manufacturers to ensure consistent results.

How can researchers optimize immunohistochemistry protocols with PRMT6 antibodies?

For optimal IHC-P staining with PRMT6 antibodies:

• Tissue preparation:

  • Use freshly fixed tissues (10% neutral buffered formalin for 24-48 hours)

  • Paraffin embed and section at 4-6μm thickness

  • Mount on positively charged slides

• Antigen retrieval (critical for nuclear antigens like PRMT6):

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Pressure cooker method: 15 minutes at full pressure

  • Alternative: microwave in citrate buffer for 20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with 5% normal serum from secondary antibody host

  • For Rabbit Polyclonal PRMT6 antibodies like ab72205, use at manufacturer-recommended dilution

  • Incubate overnight at 4°C in a humidified chamber

• Detection:

  • Use appropriate HRP/AP detection system

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount with permanent mounting medium

• Controls:

  • Always include positive control tissues (kidney and testes show high PRMT6 expression)

  • Include negative controls (omitting primary antibody)

  • Consider using PRMT6 knockdown or knockout tissues for specificity validation

Optimize incubation times and antibody concentrations for your specific tissue type, as nuclear antigens like PRMT6 may require adjusted protocols for optimal signal-to-noise ratio.

What are common issues when working with PRMT6 antibodies and their solutions?

Common challenges with PRMT6 antibody experiments and their troubleshooting approaches:

• Weak or no signal in Western blot:

  • Increase antibody concentration (try 1:250 if 1:500 recommended)

  • Extend primary antibody incubation to overnight at 4°C

  • Ensure proper sample preparation with nuclear extraction (PRMT6 is nuclear)

  • Verify protein loading with appropriate loading controls

  • Check transfer efficiency with reversible staining

• High background:

  • Increase blocking time and washing steps

  • Reduce antibody concentration

  • Use fresher antibody aliquots (avoid freeze-thaw cycles)

  • Try alternative blocking reagents (BSA vs. milk)

  • For monoclonal antibodies like 67981-1-PBS, ensure proper antibody storage at -80°C

• Multiple bands or unexpected molecular weight:

  • Confirm antibody specificity with positive controls

  • Verify sample integrity (proper lysis, protease inhibitors)

  • Check for post-translational modifications of PRMT6 (particularly automethylation at R35)

  • Use freshly prepared samples (degradation can cause multiple bands)

• Variability between experiments:

  • Standardize protocols rigorously

  • Use the same antibody lot number when possible

  • Prepare fresh buffers for each experiment

  • Maintain consistent incubation times and temperatures

Addressing these common issues will improve reproducibility and reliability of your PRMT6 antibody experiments.

How can researchers validate the specificity of their PRMT6 antibody?

To validate PRMT6 antibody specificity:

• Positive and negative controls:

  • Use cell lines with known PRMT6 expression (positive: HeLa, MCF7, NIH/3T3, Jurkat; tissue: rat testis)

  • Include PRMT6 knockdown/knockout samples as negative controls

• Pre-absorption test:

  • Pre-incubate antibody with purified PRMT6 protein or immunizing peptide

  • Compare with non-absorbed antibody (signal should be significantly reduced)

• Multiple antibody validation:

  • Test multiple PRMT6 antibodies targeting different epitopes

  • Compare staining patterns (should be consistent across antibodies)

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the PRMT6 antibody

  • Confirm pulled-down protein as PRMT6 by mass spectrometry

• Molecular weight verification:

  • PRMT6 should appear at approximately 42 kDa on Western blots

  • Verify with recombinant PRMT6 protein as a standard

• Cross-reactivity assessment:

  • Test antibody against related PRMT family members

  • Ensure specificity for PRMT6 over PRMT1-5 and PRMT7-9

Thorough validation ensures experimental reliability and data reproducibility when using PRMT6 antibodies in research applications.

How are PRMT6 monoclonal antibodies used in epigenetic research?

PRMT6 monoclonal antibodies are powerful tools in epigenetic research due to PRMT6's role in histone modification:

• Chromatin Immunoprecipitation (ChIP) applications:

  • Use PRMT6 antibodies to identify genomic regions where PRMT6 is directly bound

  • Combine with sequencing (ChIP-seq) to map genome-wide PRMT6 binding patterns

  • Correlate with histone modification marks (H3R2me2a, H3R17me2a, H3R42me2a, and H2AR26me2a)

• Co-immunoprecipitation studies:

  • Identify protein complexes associated with PRMT6 in chromatin remodeling

  • Study interactions between PRMT6 and transcription factors

• Histone methylation research:

  • Use PRMT6 antibodies alongside histone modification antibodies to study how PRMT6 generates asymmetric dimethylation modifications

  • Investigate cross-talk between H3R2 methylation by PRMT6 and other histone marks

• Transcriptional regulation studies:

  • Investigate PRMT6's role as a transcriptional coactivator for steroid hormone receptors including ESR1, ESR2, PGR, and NR3C1

  • Study effects on gene expression through reporter assays combined with PRMT6 detection

• Cellular differentiation and development research:

  • Monitor PRMT6 expression and activity during cellular differentiation

  • Correlate changes in PRMT6 localization with developmental stages

These applications rely on high-specificity antibodies that can distinguish PRMT6 from other PRMT family members in complex cellular contexts.

What is the significance of PRMT6 automethylation and how can it be studied using antibodies?

PRMT6 automethylation at residue R35 significantly impacts its stability and function, particularly its antiretroviral activity . Researchers can investigate this phenomenon using:

• Automethylation detection strategies:

  • Use liquid chromatography-mass spectrometry (LC-MS) to identify methylation at R35

  • Compare wild-type PRMT6 to R35A mutant protein in methylation assays

  • Monitor automethylation through metabolic labeling with methyl-group donors

• Stability analysis of automethylated vs. non-methylated PRMT6:

  • Research has shown that wild-type PRMT6 displays greater stability than the R35A mutant variant

  • Use cycloheximide chase assays with PRMT6 antibodies to measure protein degradation rates

  • Western blot with PRMT6 antibodies can detect different stability profiles between wild-type and R35A mutant PRMT6

• Functional impact assessment:

  • The R35A mutation that prevents automethylation impacts PRMT6's ability to inhibit HIV-1 replication

  • Use infectivity assays to measure antiviral activity while detecting PRMT6 expression levels

  • HIV-1 single-cycle TZM-bl infectivity assays have demonstrated this functional relationship

• Experimental approaches:

  • Site-directed mutagenesis to create R35A or other automethylation-deficient mutants

  • In vitro and in vivo methylation assays with recombinant proteins

  • Transient transfection of wild-type vs. mutant PRMT6 followed by stability assessments

Understanding PRMT6 automethylation provides insights into both the regulation of this enzyme and its potential therapeutic applications in HIV-1 research.

How can PRMT6 antibodies contribute to HIV-1 research?

PRMT6 antibodies are valuable tools in HIV-1 research due to PRMT6's significant antiretroviral activity:

• Mechanism studies of PRMT6-mediated viral restriction:

  • PRMT6 methylates and restricts the function of HIV proteins Tat, Rev, and Nucleocapsid (NC)

  • Use PRMT6 antibodies to detect enzyme levels in cells with varying HIV-1 susceptibility

  • Compare methylation patterns of viral proteins in the presence or absence of PRMT6

• Assessing PRMT6 stability in HIV-1 infected cells:

  • Research shows that automethylation at R35 is indispensable for PRMT6's anti-HIV-1 activity

  • Western blot with PRMT6 antibodies can monitor protein levels during infection

  • Track stability differences between wild-type and R35A mutant PRMT6 during infection cycles

• Co-localization studies:

  • Use immunofluorescence with PRMT6 antibodies to track enzyme localization during infection

  • Examine co-localization with viral proteins in different cellular compartments

• Therapeutic potential assessment:

  • Screen compounds that may enhance PRMT6 stability or activity

  • Use PRMT6 antibodies to measure protein levels in response to candidate enhancers

  • Study expression patterns in resistant vs. susceptible cell types

• Viral restriction mechanism analysis:

  • PRMT6 methylation disrupts the Tat-TAR-cyclin T1 complex and decreases Tat-specific transcriptional activation

  • It also impairs NC's ability to promote RNA annealing and initiate reverse transcription

  • PRMT6 antibodies can help characterize these interactions through co-immunoprecipitation

These applications demonstrate how PRMT6 antibodies contribute to understanding host restriction factors in HIV-1 research and potential therapeutic approaches.

What are the latest findings regarding PRMT6 in cancer biology?

PRMT6 has emerged as a significant player in cancer biology, with antibodies enabling key discoveries:

• Expression pattern analysis:

  • PRMT6 is expressed in a wide range of tissues with particularly high expression in kidney and testes

  • Altered expression patterns are observed in various cancer types

  • Antibody-based methods (IHC, Western blotting) are critical for expression profiling across tumor samples

• Epigenetic dysregulation:

  • PRMT6-mediated H3R2 methylation influences gene expression patterns in cancer cells

  • PRMT6 generates asymmetric dimethylation modifications in histones (H3R2me2a, H3R17me2a, H3R42me2a, and H2AR26me2a)

  • These modifications participate in the epigenetic regulation of oncogenes and tumor suppressors

• Interaction with transcriptional machinery:

  • PRMT6 acts as a transcriptional coactivator for several steroid hormone receptors including ESR1, ESR2, PGR, and NR3C1

  • This function may contribute to hormone-responsive cancer progression

  • Co-immunoprecipitation with PRMT6 antibodies helps characterize these interactions

• Metabolic reprogramming:

  • PRMT6 promotes fasting-induced transcriptional activation of the gluconeogenic program through methylation of the CRTC2 transcription coactivator

  • This metabolic role may influence cancer cell energy utilization

  • Antibody-based detection methods track these metabolic changes

• Potential therapeutic targeting:

  • PRMT6 inhibitors are being developed for cancer therapy

  • Antibodies are essential for validating target engagement in drug development pipelines

  • Response biomarker development relies on specific antibody detection methods

PRMT6 antibodies are indispensable tools for these cancer biology investigations, enabling both basic research and translational applications in oncology.

How should researchers optimize sample preparation for detecting low-abundance PRMT6 in primary tissues?

Detecting low-abundance PRMT6 in primary tissues requires optimized sample preparation:

• Tissue collection and preservation:

  • Process tissues immediately after collection

  • Snap-freeze in liquid nitrogen for protein analysis

  • Fix in 10% neutral buffered formalin for no more than 24 hours for IHC

  • Consider using PAXgene or other molecular fixatives that better preserve protein epitopes

• Nuclear enrichment strategies:

  • PRMT6 is predominantly nuclear , so nuclear extraction increases detection sensitivity

  • Use specialized nuclear extraction buffers with protease inhibitors

  • Consider subcellular fractionation to concentrate nuclear proteins

  • Gentle homogenization techniques prevent nuclear damage and protein loss

• Protein extraction optimization:

  • Use specialized lysis buffers containing deoxycholate for nuclear proteins

  • Include protease inhibitor cocktails to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylation states

  • Consider sonication to shear chromatin and release DNA-bound proteins

• Signal amplification methods:

  • For IHC: Use tyramide signal amplification (TSA) systems

  • For Western blot: Consider highly sensitive ECL substrates designed for low-abundance proteins

  • For immunofluorescence: Use quantum dots or other high-signal fluorophores

• Antibody selection considerations:

  • For low-abundance detection, consider polyclonal antibodies like ab72205 for their ability to recognize multiple epitopes

  • Use high-affinity monoclonal antibodies at optimized concentrations

  • Consider matching the application with the manufacturer's validation data

These optimized approaches significantly improve detection sensitivity for low-abundance PRMT6 in challenging primary tissue samples.

What are the best practices for studying PRMT6 enzyme activity rather than just protein expression?

Investigating PRMT6 enzyme activity rather than mere expression requires specialized approaches:

• Methyltransferase activity assays:

  • Immunoprecipitate PRMT6 using specific antibodies like Mouse Monoclonal PRMT6 antibody (67981-1-PBS)

  • Measure methyltransferase activity using ³H-SAM (S-adenosyl-L-methionine) as methyl donor

  • Quantify incorporation of radioactive methyl groups into substrate proteins

  • Alternative: use non-radioactive SAM analogs with coupled detection systems

• Substrate-specific methylation detection:

  • Use antibodies against methylated arginine in PRMT6 targets (H3R2me2a, H3R17me2a, etc.)

  • Compare methylation levels after PRMT6 knockdown/overexpression

  • Apply in vitro methylation assays with recombinant substrates

• Automethylation analysis:

  • Monitor PRMT6 automethylation at R35 as an indicator of enzymatic activity

  • Compare wild-type vs. catalytically inactive KLA mutant

  • Use stability assays (cycloheximide chase) to correlate automethylation with protein longevity

• Active site targeting:

  • Use active site-directed inhibitors to distinguish active vs. inactive PRMT6

  • Compare enzyme activity before and after inhibitor treatment

  • Combine with structural analysis to understand catalytic mechanism

• Enzyme kinetics:

  • Measure Km and Vmax parameters for PRMT6 with various substrates

  • Compare enzyme kinetics between wild-type and mutant versions

  • Analyze the impact of cellular conditions on enzyme activity

These approaches provide a more comprehensive understanding of PRMT6 function beyond simple expression analysis, revealing the dynamic aspects of this important epigenetic regulator.