PRMT6 Antibody

What is PRMT6 and why is it important in research?

PRMT6 is a protein arginine methyltransferase that transfers methyl groups to specific arginine residues in target proteins. It has emerged as a crucial factor in the development and progression of multiple cancer types and plays a significant role in immune regulation. PRMT6 is particularly important in research because it can inhibit antiviral innate immunity by influencing the TBK1-IRF3 signaling pathway, which mediates type-I interferon production . Research into PRMT6 provides insights into post-translational regulation mechanisms and potential therapeutic targets for immune and cancer-related diseases.

What applications are PRMT6 antibodies commonly used for?

PRMT6 antibodies are versatile research tools with multiple validated applications. Most commercial PRMT6 antibodies can be used for Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), and Flow Cytometry (FC) . Many antibodies have been validated with specific cell lines including HEK-293, HeLa, MCF-7, and SH-SY5Y cells . These applications allow researchers to detect, quantify, and localize PRMT6 in various experimental contexts.

What species reactivity should I consider when selecting a PRMT6 antibody?

Most commercially available PRMT6 antibodies show reactivity with human and mouse samples . When selecting an antibody, verify the tested and cited reactivity in the product information. For example, the 15395-1-AP antibody has been validated for reactivity with both human and mouse samples . Consider the experimental model system you are working with and ensure the antibody has been validated for your specific species to avoid false negative results.

What are the recommended dilutions for different PRMT6 antibody applications?

Optimal dilutions vary by application and specific antibody. For the 15395-1-AP antibody, the following dilutions are recommended:

It is crucial to optimize these dilutions for your specific experimental system to obtain optimal results . Titration experiments are recommended when using the antibody in a new application or cell type.

How should I perform immunoprecipitation with PRMT6 antibodies?

For immunoprecipitation of PRMT6, use approximately 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . A validated protocol involves:

• Prepare cell lysate (e.g., from SH-SY5Y cells)

• Incubate 2 μg of PRMT6 antibody with 0.35 mg lysate at an appropriate dilution (1/30 has been validated for some antibodies)

• Capture the immunocomplex using appropriate beads

• Wash thoroughly to remove non-specific binding

• Elute the precipitated proteins and analyze by Western blot

• Use a verified detection system, such as VeriBlot for IP secondary antibody (HRP)

This method has been validated for detecting endogenous PRMT6 protein interactions and can be adapted to study PRMT6's binding partners.

What controls should I include when using PRMT6 siRNA in conjunction with antibodies?

When using PRMT6 siRNA for knockdown validation experiments:

• Include at least two different PRMT6-targeting siRNAs to confirm specificity

• Always include a non-targeting control siRNA

• Validate knockdown efficiency by Western blot using your PRMT6 antibody

• Compare results between wild-type cells, non-targeting siRNA-treated cells, and multiple PRMT6 siRNA-treated samples

• Include positive control cell lines with known PRMT6 expression (e.g., HeLa, MCF7, SH-SY5Y, or LNCaP cells)

This approach provides robust validation of both the siRNA efficiency and antibody specificity.

How can I investigate PRMT6 methyltransferase activity using antibodies?

To investigate PRMT6 methyltransferase activity:

• Perform in vitro methylation assays using recombinant PRMT6 protein and 14C-labeled S-adenosyl-L-methionine (14C-SAM) as methyl donor

• Use gel electrophoresis autoradiography to detect methylated proteins

• For time-dependent methylation assays, incubate 1.5 μM PRMT6 with 10 μM 14C-SAM and quench reactions at different time points (0-120 min)

• Normalize methylation intensity data against appropriate controls (e.g., 14C-BSA)

• For PRMT6 mutant analysis, compare 5 μM of each PRMT6 mutant incubated with 30 μM 14C-SAM with or without interacting proteins like PRMT1

This methodological approach allows for quantitative assessment of PRMT6 enzymatic activity and can be used to study the impact of mutations or interacting proteins on its function.

How can I study the interaction between PRMT6 and other proteins?

To investigate protein-protein interactions involving PRMT6:

• Perform pull-down assays using tagged recombinant proteins:

  • Incubate tagged protein (e.g., MBP-tagged PRMT1) with appropriate resin (e.g., amylose beads)

  • After washing, incubate with purified PRMT6 protein

  • Wash extensively to remove unbound proteins

  • Elute complexes and analyze by Western blotting using PRMT6 antibodies

• For endogenous protein interactions:

  • Perform co-immunoprecipitation using PRMT6 antibodies

  • Use the precipitated complex to identify interacting partners by Western blot or mass spectrometry

  • Include appropriate controls (e.g., IgG control, PRMT6-depleted samples)

• For in-cell validation:

  • Perform proximity ligation assays or fluorescence resonance energy transfer (FRET) using PRMT6 antibodies and antibodies against potential interacting partners

These methods provide complementary approaches to validate and characterize PRMT6 protein interactions.

What approaches can I use to study PRMT6's role in antiviral immunity?

To investigate PRMT6's role in antiviral immunity:

• Generate PRMT6-deficient cell lines or use tissues from PRMT6-knockout mice

• Challenge with viruses (e.g., VSV, HSV-1) and assess:

  • Viral replication by measuring viral RNA levels using qPCR

  • Type-I interferon production (IFN-α, IFN-β) by ELISA or qPCR

  • Activation of the TBK1-IRF3 signaling pathway by co-immunoprecipitation and Western blot

  • Inflammatory cytokine production (e.g., IL-6)

• Perform mechanistic studies:

  • Assess PRMT6-IRF3 binding upon viral infection

  • Determine if PRMT6 inhibits TBK1-IRF3 interaction

  • Investigate whether the effect is dependent or independent of PRMT6 methyltransferase activity

These approaches help elucidate PRMT6's functional role in regulating antiviral immune responses.

What are common challenges in PRMT6 immunohistochemistry and how can I overcome them?

Common challenges in PRMT6 IHC include weak signal, high background, and variability between tissues. To address these issues:

• Optimize antigen retrieval:

  • Use TE buffer at pH 9.0 (recommended for many PRMT6 antibodies)

  • Alternatively, try citrate buffer at pH 6.0 if optimal results aren't achieved with TE buffer

• Adjust antibody concentration:

  • Start with the recommended dilution range (1:50-1:500 for many antibodies)

  • Perform titration experiments to determine optimal concentration for your specific tissue

• Validate on appropriate tissues:

  • Human prostate cancer tissue, kidney tissue, breast cancer tissue, and testis tissue have shown positive PRMT6 staining

  • Include positive control tissues in each experiment

• If background is high:

  • Extend blocking steps

  • Include additional washing steps

  • Consider using more specific detection systems

These optimizations can significantly improve the quality and reproducibility of PRMT6 IHC staining.

How can I troubleshoot unexpected bands in Western blot for PRMT6?

When troubleshooting Western blot issues with PRMT6 antibodies:

• Verify the expected band size:

  • PRMT6 has a calculated molecular weight of 41-42 kDa

  • Confirm on positive control samples (e.g., HeLa, MCF-7, or HEK-293 cells)

• If observing multiple bands:

  • Check for post-translational modifications of PRMT6

  • Verify specificity using PRMT6 knockdown samples (siRNA treated cells)

  • Optimize antibody dilution (try 1:2000-1:10000 range)

  • Extend blocking and washing steps to reduce non-specific binding

• For weak or no signal:

  • Increase protein loading

  • Decrease antibody dilution

  • Extend exposure time

  • Verify PRMT6 expression in your cell type or tissue

• Include validated controls:

  • Positive control: Lysates from cells known to express PRMT6 (HEK-293, HeLa, MCF-7)

  • Negative control: PRMT6 knockdown samples

These systematic approaches can help resolve Western blot issues when working with PRMT6 antibodies.

How does viral infection affect PRMT6 expression, and what are the implications for research?

Viral infection significantly impacts PRMT6 expression:

• Multiple viruses upregulate PRMT6 protein levels:

  • VSV and HSV-1 infection markedly increases PRMT6 expression in mouse primary peritoneal macrophages and human THP-1 cells

  • VSV infection also increases PRMT6 in human A549 cells

  • HBV transfection increases PRMT6 expression in HepG2.2.15 cells compared to parental HepG2 cells

• Interestingly, PRMT6 mRNA levels are not significantly altered upon viral infection, suggesting post-transcriptional regulation

• The increased PRMT6 expression negatively regulates antiviral innate immunity by:

  • Inhibiting the TBK1-IRF3 interaction

  • Reducing IRF3 activation

  • Decreasing type-I interferon production

These findings suggest PRMT6 may be upregulated as a viral evasion strategy to dampen host immune responses. When designing experiments to study antiviral responses, researchers should consider monitoring PRMT6 levels as a potential regulatory factor.

What is known about the interaction between PRMT1 and PRMT6, and how can this be studied?

The interaction between PRMT1 and PRMT6 represents an important research area:

• PRMT6 has been identified as a novel substrate of PRMT1:

  • PRMT1 can methylate PRMT6 at specific arginine residues

  • This interaction can be studied using in vitro methylation assays with recombinant proteins and 14C-SAM

• To study this interaction:

  • Perform pull-down assays with purified proteins

  • Use liquid chromatography-tandem mass spectrometry to identify specific methylation sites

  • Generate site-specific mutations (e.g., PRMT6-M166A) to assess the functional importance of specific residues

  • Compare kinetic parameters of wild-type and mutant proteins

• Functional implications:

  • This interaction may represent a regulatory mechanism between different PRMT family members

  • The methylation of PRMT6 by PRMT1 could affect its activity, stability, or interactions with other proteins

Understanding these interactions provides insight into the complex regulatory networks involving PRMTs in cellular functions.

What is the significance of PRMT6 in antiviral immunity and how does PRMT6 deficiency affect immune responses?

PRMT6 plays a significant role in regulating antiviral immunity:

• PRMT6 functions as a negative regulator of antiviral innate immunity:

  • It inhibits the TBK1-IRF3 signaling pathway

  • Viral infection enhances PRMT6 binding to IRF3

  • This inhibits the interaction between IRF3 and TBK1, reducing type-I interferon production

• PRMT6-deficient mice exhibit enhanced antiviral immunity:

  • Reduced viral titers in various organs 18 hours post-infection

  • Decreased viral RNA levels in infected organs

  • Less inflammatory cell infiltration in the lungs

  • Significantly higher production of IFN-α, IFN-β, and IL-6 in response to viral infection

• Interestingly, this regulatory function of PRMT6 appears to be independent of its methyltransferase activity, suggesting a non-canonical role

• The physiological significance may be two-fold:

  • PRMT6 may protect the host from damaging effects of an overactive immune system

  • Viruses may exploit PRMT6 upregulation as a mechanism to escape immune detection

These findings highlight PRMT6 as a potential therapeutic target for enhancing antiviral immunity or treating conditions characterized by excessive interferon production.