PI4KG8 Antibody

What is PI4KG8 in Arabidopsis thaliana and why study it with antibodies?

PI4KG8 (Phosphatidylinositol 4-Kinase Gamma 8) in Arabidopsis thaliana is part of the PI4K family involved in phosphoinositide signaling pathways. Antibodies against this protein allow researchers to study its expression patterns, localization, and functional relationships in plant cellular processes. Using the recombinant protein-raised antibody (CSB-PA998836XA01DOA) enables specific detection of this target in plant tissues, providing insights into phosphoinositide metabolism that regulates membrane trafficking, cytoskeletal organization, and stress responses in plants .

What validated applications can PI4KG8 Antibody be used for?

The PI4KG8 Antibody (CSB-PA998836XA01DOA) has been validated specifically for ELISA and Western Blot applications in Arabidopsis thaliana research. These techniques allow quantitative assessment of protein expression levels (ELISA) and molecular weight confirmation/expression analysis (Western Blot). Unlike therapeutic antibodies that require more extensive validation across multiple platforms, this research-grade antibody has been optimized for these specific applications to ensure reliable identification of the antigen .

What are the optimal storage conditions for maintaining PI4KG8 antibody activity?

PI4KG8 Antibody should be stored immediately upon receipt at either -20°C or -80°C. Crucially, repeated freeze-thaw cycles must be avoided as they can cause antibody degradation through conformational changes in the antibody structure, particularly in the variable regions that determine antigen specificity. The antibody is supplied in liquid form containing 0.03% Proclin 300 preservative, which helps maintain stability during proper storage. For long-term studies spanning several months, consider aliquoting the antibody into single-use volumes to minimize freeze-thaw damage .

How should western blot protocols be optimized for PI4KG8 Antibody?

When optimizing western blot protocols for PI4KG8 Antibody, begin with standard conditions but be prepared to adjust several parameters. First, ensure complete protein extraction from Arabidopsis tissue using a buffer containing phosphatase inhibitors to preserve phosphorylation states. For SDS-PAGE, use 8-10% gels to effectively resolve higher molecular weight proteins like kinases. During transfer, use PVDF membranes rather than nitrocellulose for better protein retention. For blocking, 5% BSA often works better than milk for phosphoprotein detection. Start with a 1:1000 antibody dilution in overnight 4°C incubation, then adjust concentration if necessary. To improve signal-to-noise ratio, incorporate stringent washing steps (4-5 washes of 10 minutes each) with TBS-T (0.1% Tween-20) .

What controls are essential when using PI4KG8 Antibody in immunological assays?

When designing experiments with PI4KG8 Antibody, implementation of appropriate controls is critical for result validation. Always include a negative control using wild-type Arabidopsis tissue alongside samples from PI4KG8 knockout/knockdown lines to confirm specificity. Additionally, use a positive control with recombinant PI4KG8 protein at known concentrations to establish detection limits. For blocking experiments, pre-incubate antibody with excess immunizing peptide to confirm binding specificity, similar to epitope-blocking validation approaches used with other antibodies . When multiple antibodies are used simultaneously (e.g., in co-localization studies), include single-antibody controls to assess potential cross-reactivity. These systematic controls help discriminate between specific signals and experimental artifacts .

How can I determine the optimal working dilution for PI4KG8 Antibody in ELISA assays?

To determine the optimal working dilution for PI4KG8 Antibody in ELISA, perform a systematic titration experiment. Prepare a dilution series (typically 1:500, 1:1000, 1:2000, 1:5000, and 1:10000) of the antibody while keeping antigen concentration constant across wells. Plot the resulting signal intensity against antibody dilution to identify the concentration that provides maximal specific signal with minimal background. The optimal dilution typically falls in the steep linear portion of the curve, before signal saturation occurs. Additionally, test different blocking agents (BSA, milk protein, or commercial blockers) to minimize background. This optimization approach, analogous to methods used for other research antibodies, ensures both sensitivity and specificity in your ELISA applications .

How can PI4KG8 Antibody be used for subcellular localization studies in Arabidopsis?

For subcellular localization studies of PI4KG8 in Arabidopsis using immunofluorescence, first optimize tissue fixation (typically 4% paraformaldehyde) and permeabilization conditions to maintain cellular architecture while allowing antibody access. The CSB-PA998836XA01DOA antibody, though not explicitly validated for immunofluorescence, can potentially be applied following protocols similar to those used for other plant protein kinases. Begin with a 1:100-1:500 dilution range for primary antibody incubation. Co-stain with established organelle markers (e.g., ER, Golgi, plasma membrane) to assess co-localization. Confocal microscopy with Z-stack acquisition will provide three-dimensional localization data. Validate all observations using PI4KG8 knockout/knockdown plants as negative controls to confirm staining specificity .

What strategies can improve PI4KG8 Antibody penetration in thick plant tissues?

Improving antibody penetration in thick plant tissues requires specialized techniques beyond standard protocols. For PI4KG8 detection in mature Arabidopsis tissues, implement extended fixation times (12-24 hours) with gentle agitation to ensure complete penetration. Use vacuum infiltration cycles (5 minutes vacuum, 1 minute release, repeated 3-5 times) to remove air from intercellular spaces. Increase permeabilization by incorporating 0.1-0.3% Triton X-100 in all buffers and extending incubation times. For very thick tissues, consider physical sectioning (50-100 μm vibratome sections) prior to immunostaining. Employing these techniques can significantly enhance antibody accessibility to intracellular targets while preserving tissue morphology, similar to approaches used for other plant tissue immunostaining procedures .

How might phosphorylation states affect PI4KG8 detection by this antibody?

Phosphorylation states may significantly impact PI4KG8 detection by this antibody, particularly if the epitope region contains phosphorylation sites. Kinases like PI4KG8 often undergo regulatory phosphorylation that can alter protein conformation and potentially mask or expose antibody binding sites. To assess this potential variable, compare antibody recognition under different conditions that alter phosphorylation states: (1) treat samples with phosphatase inhibitors versus lambda phosphatase treatment, (2) compare samples from plants under normal conditions versus stress conditions known to activate kinase pathways, and (3) analyze samples with phospho-specific antibodies alongside this PI4KG8 antibody. These comparative analyses will reveal whether the antibody (CSB-PA998836XA01DOA) exhibits phospho-dependent recognition bias, which is critical for accurate interpretation of experimental results .

What are the most common causes of weak or absent signal when using PI4KG8 Antibody?

When encountering weak or absent signals with PI4KG8 Antibody, systematically investigate several potential causes. First, verify antibody viability; excessive freeze-thaw cycles or improper storage temperatures can degrade activity. Second, check protein extraction efficiency, as membrane-associated kinases like PI4KG8 may require specialized extraction buffers containing appropriate detergents. Third, assess protein transfer efficiency during western blotting by using reversible stains like Ponceau S. Fourth, optimize incubation conditions—try extending primary antibody incubation to overnight at 4°C rather than 1-2 hours at room temperature. Fifth, enhance detection sensitivity by using amplification systems like biotin-streptavidin or try increasing antibody concentration (1:500 instead of 1:1000). Finally, verify that your experimental conditions haven't downregulated the target protein expression below detection limits .

How can I distinguish between specific and non-specific bands in Western blots using PI4KG8 Antibody?

Distinguishing between specific and non-specific bands requires multiple validation strategies. First, compare band patterns between wild-type and PI4KG8 knockout/knockdown Arabidopsis samples; specific bands should be absent or significantly reduced in knockdown samples. Second, perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide before application; specific bands should disappear while non-specific bands remain. Third, validate molecular weight—PI4KG8 should appear at its predicted size, though post-translational modifications may cause slight shifts. Fourth, try alternative detection methods like mass spectrometry to confirm protein identity. Finally, compare results across different tissue types or developmental stages where PI4KG8 expression varies, as specific bands should show biologically relevant expression patterns while non-specific bands typically remain constant .

What strategies can resolve cross-reactivity issues with PI4KG8 Antibody in Arabidopsis research?

Resolving cross-reactivity issues with PI4KG8 Antibody requires a multi-faceted approach. First, increase washing stringency by using higher salt concentration buffers (up to 500mM NaCl in TBS-T) to disrupt low-affinity non-specific interactions. Second, optimize blocking conditions by testing different agents (BSA, casein, commercial blockers) at various concentrations (3-5%) and extended blocking times (2-3 hours). Third, reduce primary antibody concentration incrementally to find the minimum effective concentration that maintains specific signal while reducing non-specific binding. Fourth, add low concentrations (0.1-0.5%) of non-ionic detergents like Tween-20 to all antibody dilution buffers. Fifth, pre-absorb the antibody with Arabidopsis tissue lysate from PI4KG8 knockout plants to remove antibodies that bind to other proteins. These approaches, similar to cross-reactivity resolution strategies for other plant antibodies, can significantly improve signal specificity .

How does the specificity of recombinant-raised PI4KG8 Antibody compare to synthetic peptide-raised antibodies?

The PI4KG8 Antibody (CSB-PA998836XA01DOA) was raised against recombinant Arabidopsis thaliana PI4KG8 protein, which typically offers different epitope recognition patterns compared to synthetic peptide-raised antibodies. Recombinant protein-raised antibodies generally recognize multiple epitopes across the protein's surface, potentially providing more robust detection across different experimental conditions. This polyclonal nature enables continued detection even if some epitopes become modified or obstructed. In contrast, synthetic peptide antibodies target specific linear sequences, offering precise epitope targeting but potentially reduced detection if that region undergoes conformational changes or post-translational modifications. When designing experiments, consider that this recombinant-raised antibody likely provides broader epitope recognition, making it suitable for diverse applications where comprehensive protein detection is required .

What considerations are important when using PI4KG8 Antibody for quantitative analyses?

For quantitative analysis using PI4KG8 Antibody, several critical factors must be addressed to ensure reliable results. First, establish a standard curve using purified recombinant PI4KG8 protein to determine the linear detection range. Second, normalize target protein measurements to appropriate loading controls (e.g., actin, tubulin, or GAPDH) that remain stable under your experimental conditions. Third, validate antibody lot-to-lot consistency by comparing standard curves between antibody lots. Fourth, optimize sample preparation to ensure complete and consistent protein extraction across all experimental groups. Fifth, implement technical replicates (minimum of three) and biological replicates (different plants/experiments) to establish statistical validity. Finally, consider using absolute quantification methods like AQUA (Absolute Quantification) peptides as internal standards for mass spectrometry validation of antibody-based quantifications .

How can PI4KG8 Antibody be used to study protein-protein interactions in Arabidopsis signaling pathways?

PI4KG8 Antibody can be instrumental in studying protein-protein interactions within Arabidopsis signaling networks through several advanced approaches. First, use co-immunoprecipitation (co-IP) by immobilizing the PI4KG8 antibody to precipitation matrices (protein A/G beads) to capture PI4KG8 along with its interacting partners, which can then be identified by mass spectrometry. Second, implement proximity ligation assays (PLA) where the PI4KG8 antibody is used alongside antibodies against suspected interaction partners, generating fluorescent signals only when proteins are in close proximity (<40nm). Third, utilize the antibody in ChIP-seq experiments if PI4KG8 has nuclear localization to identify potential DNA-binding activities. Fourth, apply FRET-FLIM microscopy using the antibody to validate interactions observed through other methods. These approaches collectively provide complementary evidence for functional protein networks involving PI4KG8 in phosphoinositide signaling pathways .