Phospho-PLCG2 (Tyr753) Antibody

What is PLCG2 and what is the significance of its phosphorylation at Tyr753?

Phospholipase C-gamma-2 (PLCG2) is a crucial enzyme in cell signaling that plays a vital role in the initiation of receptor-mediated signal transduction. It functions by generating two second messengers: inositol 1,4,5-triphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate . PLCG2 belongs to a family of mammalian PLC isozymes that includes various subtypes such as PLC beta1-4, PLC gamma1-2, PLC delta1-2, and PLCe . Phosphorylation at tyrosine 753 (Tyr753) represents a key regulatory post-translational modification that modulates PLCG2 activity, affecting downstream signaling cascades particularly in immune and cardiovascular systems . This specific phosphorylation site is often used as a biomarker for PLCG2 activation in various cellular contexts and experimental models, making antibodies against this modification valuable tools for investigating signal transduction pathways.

What are the common applications for Phospho-PLCG2 (Tyr753) antibody?

Phospho-PLCG2 (Tyr753) antibodies are utilized across multiple experimental platforms:

These applications allow researchers to investigate PLCG2 signaling in various contexts, including normal cellular processes and disease states. When selecting an application, consideration should be given to the level of sensitivity required, the nature of the starting material, and the specific research question being addressed.

What are the key characteristics of commercially available Phospho-PLCG2 (Tyr753) antibodies?

Most commercially available Phospho-PLCG2 (Tyr753) antibodies share several important characteristics:

Understanding these characteristics is essential for proper experimental design, particularly when planning multi-species studies or when specific detection parameters are required.

What are the recommended storage and handling conditions for Phospho-PLCG2 (Tyr753) antibodies?

Optimal preservation of antibody activity requires specific storage and handling protocols:

Most manufacturers recommend storing Phospho-PLCG2 (Tyr753) antibodies at -20°C for long-term preservation or at 4°C for short-term use . The antibodies are typically supplied in a stabilizing buffer containing phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain antibody integrity and prevent microbial contamination. It is advisable to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of activity . When handling the antibody, always use sterile technique, and consider preparing working aliquots to minimize freeze-thaw events. For maximum sensitivity in critical experiments, freshly thawed aliquots are recommended. Always centrifuge the antibody vial briefly before opening to collect all liquid at the bottom of the tube.

How can researchers validate the specificity of Phospho-PLCG2 (Tyr753) antibodies?

Validation is a critical step to ensure experimental rigor:

A comprehensive validation approach includes several complementary techniques. First, perform Western blot analysis comparing samples treated with phosphatase versus untreated controls to confirm phospho-specificity. Second, conduct experiments with stimulation conditions known to induce PLCG2 Tyr753 phosphorylation alongside appropriate negative controls. Third, consider using PLCG2 knockout or knockdown models as negative controls . The antibodies are specifically designed to detect endogenous levels of PLCG2 only when phosphorylated at tyrosine 753, and not the unphosphorylated form . Additionally, peptide competition assays using the immunizing phosphopeptide (S-L-Y(p)-D-V) can provide further confirmation of specificity . For advanced validation, consider using targeted mass spectrometry to correlate antibody-based detection with direct peptide identification.

What controls should be included when designing experiments using Phospho-PLCG2 (Tyr753) antibodies?

Robust experimental design requires appropriate controls:

When analyzing phosphorylation dynamics, time-course experiments with appropriate stimulation conditions are also recommended. For immunofluorescence applications, include counterstains to verify subcellular localization patterns. These controls collectively ensure that observed signals are specific to phosphorylated PLCG2 at Tyr753.

How should Western blot protocols be optimized for Phospho-PLCG2 (Tyr753) detection?

Western blot optimization requires attention to several key parameters:

When detecting Phospho-PLCG2 (Tyr753), cell lysis should be performed using buffers containing phosphatase inhibitors to preserve phosphorylation status . A recommended dilution range of 1:500-1:1000 has been established for Western blot applications . For optimal results, use freshly prepared samples and consider shorter transfer times for large proteins like PLCG2 (approximately 150 kDa). For detection, high-sensitivity chemiluminescence substrates are often preferred due to the potentially low abundance of phosphorylated species. Membrane blocking should utilize BSA rather than milk, as milk contains phosphoproteins that may interfere with detection. Following transfer, overnight primary antibody incubation at 4°C generally yields better results than shorter incubations at room temperature. For quantitative analysis, always perform parallel blots for total PLCG2 to calculate phosphorylation/total protein ratios.

What are the best sample preparation methods for preserving PLCG2 phosphorylation status?

Maintaining phosphorylation during sample preparation is critical:

To preserve the phosphorylation status at Tyr753, samples should be processed rapidly and kept cold throughout preparation. Lysis buffers must contain both phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and sodium pyrophosphate) and protease inhibitors . For tissue samples, snap freezing in liquid nitrogen immediately after collection is essential. When working with cultured cells, direct lysis in hot SDS sample buffer can better preserve transient phosphorylation events compared to conventional lysis methods. For immunoprecipitation applications, use non-denaturing lysis buffers supplemented with phosphatase inhibitors. If analysis cannot be performed immediately, store lysates at -80°C in single-use aliquots. For immunohistochemistry, phospho-epitopes are best preserved with phosphate-free fixatives, and antigen retrieval methods should be carefully optimized.

How can the AlphaLISA SureFire Ultra assay be optimized for Phospho-PLCG2 (Tyr753) detection?

The AlphaLISA SureFire Ultra assay provides a sensitive quantitative approach:

This sandwich immunoassay technology offers a highly sensitive method for detecting phosphorylated PLCG2 at Tyr753 in cellular lysates . For optimal results, sample preparation should follow manufacturer's protocols precisely, using 10 μL sample volumes as recommended . The assay utilizes a donor bead coated with streptavidin to capture a biotinylated antibody, while acceptor beads coated with CaptSure™ agent immobilize a second antibody labeled with a CaptSure tag . When phosphorylated PLCG2 is present, the two antibodies bring donor and acceptor beads into proximity, generating a quantifiable signal proportional to protein concentration . To maximize sensitivity, perform the assay in low-light conditions, as the Alpha (Amplified Luminescent Proximity Homogeneous Assay) donor beads are light-sensitive. Allow all components to equilibrate to room temperature before use, and include standard curves with known quantities of phosphorylated protein for accurate quantification.

How does PLCG2 Tyr753 phosphorylation relate to disease mechanisms?

PLCG2 phosphorylation has significant implications in disease contexts:

PLCG2 signaling plays crucial roles in multiple physiological systems, with particularly important functions in immune and cardiovascular contexts . Aberrant phosphorylation of PLCG2 at Tyr753 has been implicated in several pathological conditions. In immune disorders, dysregulated PLCG2 phosphorylation can contribute to autoimmune pathogenesis, making it a target of interest in conditions like Familial Cold Autoinflammatory Syndrome 3 (FCAS3) . The acronym APLAID (Autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation) underscores the connection between PLCG2 dysfunction and immune disorders . In cardiovascular research, PLCG2 has emerged as an area of interest due to its role in signaling cascades that influence cardiac function and vascular homeostasis . Additionally, abnormal accumulation of PLC delta (a related family member) has been observed in autopsied brains with Alzheimer's disease, suggesting potential roles for phospholipase C signaling in neurodegenerative processes .

What are the advantages and limitations of using polyclonal versus monoclonal antibodies for Phospho-PLCG2 (Tyr753) detection?

Understanding antibody characteristics informs optimal selection:

How can multiplexed approaches be used to study PLCG2 phosphorylation in signaling networks?

Multiplexed detection allows comprehensive pathway analysis:

To understand PLCG2 signaling in broader network contexts, researchers can employ several multiplexing strategies. For Western blot applications, sequential reprobing of membranes with antibodies against different phosphorylation sites or related signaling molecules is possible after thorough stripping. Alternatively, multiplex fluorescent Western blotting using spectrally distinct secondary antibodies can simultaneously detect multiple targets. For microscopy, multi-color immunofluorescence with appropriate controls for antibody cross-reactivity allows visualization of spatial relationships between phosphorylated PLCG2 and other signaling components. The AlphaLISA SureFire Ultra platform offers multiplex capabilities that can be leveraged to simultaneously measure phosphorylated PLCG2 and related signaling molecules . Flow cytometry-based phospho-protein detection provides another powerful approach for examining heterogeneity in PLCG2 activation at the single-cell level while simultaneously assessing other cellular parameters.

What are common issues encountered when using Phospho-PLCG2 (Tyr753) antibodies and how can they be resolved?

Anticipating and resolving technical challenges improves experimental outcomes:

When working with phospho-specific antibodies like Phospho-PLCG2 (Tyr753), timing of cell lysis following stimulation is critical, as phosphorylation can be transient. For reproducible results, standardize all aspects of sample preparation, including cell culture conditions, stimulation protocols, and lysis procedures.

How can Phospho-PLCG2 (Tyr753) antibodies be used effectively in immunohistochemistry applications?

Successful immunohistochemistry requires specific technical considerations:

While the search results primarily reference Western blot and immunofluorescence applications, immunohistochemical analysis of Phospho-PLCG2 (Tyr753) has been documented in human lymph node tissue . For optimal results in FFPE (formalin-fixed, paraffin-embedded) tissues, antigen retrieval is critical, with citrate buffer (pH 6.0) often providing good results for phospho-epitopes. Endogenous phosphatase activity in tissues necessitates thorough blocking with specific phosphatase inhibitors during sample preparation and staining. Dilution ratios around 1:100 may serve as a starting point but should be optimized for each tissue type and fixation method . Positive controls using tissues known to contain activated PLCG2 (such as lymphoid tissues) are essential for protocol validation . For chromogenic detection, avoid phosphate-based buffers during the color development step to prevent interference with phospho-epitope detection. When interpreting results, consider that phosphorylation status can vary significantly across different regions within the same tissue sample.