PPM1G Antibody

What is PPM1G and why is it significant in research?

PPM1G (also known as PP2Cγ and PP2CG) is a member of the PP2C family of serine/threonine phosphatases. It plays crucial roles in multiple cellular processes including protein translation regulation and innate immune response modulation. As a magnesium/manganese-dependent phosphatase, PPM1G has gained significant research interest due to its involvement in the dephosphorylation of key regulatory proteins like 4E-BP1, which controls cap-dependent protein translation . Additionally, recent research has identified PPM1G as a negative regulator of innate immune pathways mediated by STING and MAVS, highlighting its importance in viral immune evasion mechanisms .

Which species does the PPM1G antibody detect and what is its reactivity profile?

The PPM1G antibody (15532-1-AP) demonstrates confirmed reactivity with human, mouse, and rat samples. Published literature has primarily focused on human applications, but validation data shows cross-reactivity across these mammalian models. The antibody recognizes the full protein phosphatase 1G (formerly 2C), magnesium-dependent, gamma isoform, with a calculated molecular weight of 59 kDa, though it is typically observed at approximately 70 kDa in experimental applications .

What are the standard storage conditions for maintaining PPM1G antibody activity?

For optimal preservation of antibody activity, PPM1G antibody should be stored at -20°C in its supplied buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3). Under these conditions, the antibody remains stable for one year after shipment. While aliquoting is generally recommended for antibody storage, it is noted as unnecessary for this particular antibody when stored at -20°C. Smaller volume formulations (20μl) contain 0.1% BSA as a stabilizing agent .

What are the validated applications for PPM1G antibody and their recommended dilutions?

The PPM1G antibody (15532-1-AP) has been validated for multiple experimental applications with specific recommended dilution ranges:

These recommendations serve as starting points, and the manufacturer advises titrating the antibody in each specific testing system to achieve optimal results. Application success may be sample-dependent, and researchers should consult validation data galleries for specific tissue or cell types .

What is the optimal protocol for PPM1G detection in Western blotting?

For Western blot detection of PPM1G, researchers should first prepare protein lysates from appropriate samples (validated positive samples include HEK-293T cells, mouse lung tissue, Jurkat cells, and MCF-7 cells). After standard SDS-PAGE separation and transfer to membrane, block using standard blocking buffer and incubate with PPM1G antibody at a dilution between 1:500-1:3000 (start with 1:1000 for optimization). The antibody will detect a band at approximately 70 kDa, which differs slightly from the calculated molecular weight of 59 kDa—this discrepancy is common and likely due to post-translational modifications. Published studies have successfully used this antibody to monitor PPM1G expression levels in knockdown experiments and protein-protein interaction studies .

How should antigen retrieval be performed for IHC detection of PPM1G?

For immunohistochemical detection of PPM1G, antigen retrieval is a critical step that significantly impacts staining quality. The recommended protocol involves antigen retrieval with TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 can be used, though potentially with different retrieval efficiency. The antibody has been successfully validated on multiple tissue types including mouse kidney, testis, and spleen tissues, as well as human heart, kidney, skin, placenta, brain, and liver tissues. For optimal results, use the recommended dilution range of 1:50-1:500, with initial testing at 1:200 recommended for most fixed tissue specimens .

How does PPM1G regulate protein translation and what experimental approaches confirm this?

PPM1G regulates cap-dependent protein translation by controlling the phosphorylation state of 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1). Research has demonstrated that PPM1G directly binds to and dephosphorylates 4E-BP1 at key phosphorylation sites (Thr-37/46 and Ser-65). This regulatory function has been confirmed through several experimental approaches:

• Co-immunoprecipitation studies showing direct interaction between PPM1G and 4E-BP1

• In vitro dephosphorylation assays with purified PPM1G acting on phosphorylated 4E-BP1

• PPM1G knockdown experiments showing increased 4E-BP1 phosphorylation

• Measurement of cap-dependent translation using dual luciferase reporter systems in control versus PPM1G knockdown cells

These findings collectively establish PPM1G as a negative regulator of protein translation. Knockdown of PPM1G results in increased protein synthesis and cell growth, which can be reversed by expressing a phosphorylation-deficient 4E-BP1 mutant (4E-BP1-4A) .

What role does PPM1G play in innate immune response regulation?

Recent research has identified PPM1G as a critical negative regulator of innate immune signaling pathways. Specifically, PPM1G restricts both cytosolic DNA and RNA sensing pathways by:

• Dephosphorylating activated STING (stimulator of interferon genes) in the DNA sensing pathway

• Dephosphorylating MAVS (mitochondrial antiviral signaling protein) in the RNA sensing pathway

This dual regulatory function positions PPM1G as a natural balancer of antiviral responses. Interestingly, this regulatory mechanism appears to be exploited by certain viruses, particularly Kaposi's sarcoma-associated herpesvirus (KSHV). The KSHV tegument protein ORF33 has been shown to interact with STING/MAVS and enhance the recruitment of PPM1G to these complexes, thereby promoting dephosphorylation and suppressing antiviral signaling .

How can PPM1G antibody be used to study viral immune evasion mechanisms?

The PPM1G antibody provides researchers with a valuable tool for investigating viral immune evasion strategies. Experimental approaches include:

• Co-immunoprecipitation studies to detect interactions between PPM1G and viral proteins

• Western blotting to assess PPM1G recruitment to immune signaling complexes during viral infection

• Immunofluorescence microscopy to visualize PPM1G localization changes in infected versus uninfected cells

• Combining PPM1G knockdown with viral infection assays to assess changes in antiviral response

Research has shown that inhibition of PPM1G expression improves antiviral responses against both DNA and RNA viruses, suggesting that targeting PPM1G could potentially enhance innate immunity during viral infections .

Why might PPM1G antibody detection show discrepancies between calculated and observed molecular weights?

The PPM1G antibody detects a protein band at approximately 70 kDa, which differs from the calculated molecular weight of 59 kDa. This discrepancy is not uncommon in protein detection and may result from:

• Post-translational modifications like phosphorylation, glycosylation, or ubiquitination

• Alternative splicing generating different isoforms

• The highly charged nature of phosphatases affecting their migration in SDS-PAGE

To confirm specificity, researchers should include appropriate controls such as PPM1G knockdown samples or competition assays with immunizing peptide. The observed molecular weight has been consistently reported across multiple studies and cell types, suggesting this is the genuine migration pattern of the protein rather than non-specific binding .

How can researchers efficiently validate PPM1G knockdown or knockout for functional studies?

Validation of successful PPM1G manipulation is crucial for interpreting functional studies. A comprehensive validation approach includes:

• Western blot analysis using the PPM1G antibody (15532-1-AP) at 1:1000 dilution to confirm protein reduction

• qRT-PCR to verify mRNA reduction (independent of antibody detection)

• Functional validation by measuring increased phosphorylation of known PPM1G substrates (particularly 4E-BP1 at Thr-37/46 and Ser-65 sites)

• Rescue experiments by re-expressing shRNA-resistant PPM1G to confirm phenotype specificity

Published studies have successfully used lentiviral shRNA systems with at least two different targeting sequences to establish specific PPM1G knockdown. For experimental consistency, researchers should maintain stable knockdown cell lines and periodically revalidate PPM1G reduction .

What are the key considerations when designing co-immunoprecipitation experiments to study PPM1G interactions?

PPM1G interaction studies through co-immunoprecipitation require careful consideration of several factors:

• Choice of lysis buffer: Use buffers that maintain phosphatase activity (typically containing Mn²⁺ or Mg²⁺) but include phosphatase inhibitors when preserving substrate phosphorylation status is desired

• Antibody orientation: Both antibody configurations have been successfully used - immunoprecipitating PPM1G to detect binding partners or immunoprecipitating suspected interacting proteins to detect PPM1G

• Detection conditions: When blotting for PPM1G in co-IPs, use the antibody at 1:1000 dilution for optimal detection

• Controls: Include IgG control immunoprecipitations and, where possible, knockdown controls

Research has demonstrated that PPM1G interactions with substrates like 4E-BP1 occur independently of their phosphorylation status, suggesting stable enzyme-substrate interactions rather than transient catalytic interactions .

How do the experimental outcomes of PPM1G studies differ between cell types and physiological contexts?

When studying PPM1G functions across different cell types, researchers should consider several context-dependent variables:

• Basal expression levels: PPM1G expression varies across tissue types, with detectable expression in multiple human and mouse tissues

• Substrate availability: The presence and abundance of key substrates (e.g., 4E-BP1, STING, MAVS) may differ between cell types

• Signaling pathway activity: The relative importance of PPM1G-regulated pathways varies by cell type and physiological state

Studies have successfully examined PPM1G function in multiple cell lines including HEK-293E, HCT116 colon cancer cells, and various immune cells. The antibody has demonstrated reliable detection across these contexts, though optimal dilutions may require adjustment. For translational research, validation in primary cells or in vivo models is recommended as findings from immortalized cell lines may not fully recapitulate physiological functions .

How can PPM1G antibody be applied to study its role in antiviral immunity?

PPM1G has recently been identified as a negative regulator of innate immune pathways, opening new research applications for the PPM1G antibody. Researchers can use this antibody to:

• Track PPM1G recruitment to immune signaling complexes during viral infection using immunoprecipitation and Western blotting

• Assess changes in PPM1G localization during immune activation using immunofluorescence microscopy

• Evaluate PPM1G expression levels in different immune cell types and their response to pathogen stimuli

• Investigate whether viruses modulate PPM1G expression or activity as an immune evasion strategy

Experimental evidence indicates that inhibition of PPM1G expression enhances antiviral responses against both DNA and RNA viruses, suggesting PPM1G as a potential therapeutic target for boosting immunity during viral infections .

What methodological approaches can resolve contradictory findings about PPM1G functions?

As with many multifunctional proteins, research on PPM1G sometimes yields apparently contradictory results. To resolve such discrepancies, researchers should consider:

• Cell type specificity: Using the PPM1G antibody to compare expression and localization across multiple cell types

• Catalytic versus scaffolding functions: Distinguishing between enzymatic activity and protein-protein interaction roles

• Context-dependent regulation: Examining PPM1G function under different stimuli and stresses

• Substrate specificity: Developing comprehensive substrate profiles through phosphoproteomic analysis

The PPM1G antibody can be employed in substrate-trapping experiments, where catalytically inactive mutants of PPM1G are used to capture substrates for subsequent identification by mass spectrometry. This approach has proven valuable for identifying novel phosphatase substrates and may reveal additional PPM1G functions .