PSAT1 Antibody, FITC conjugated

What is PSAT1 and what cellular functions does it perform?

PSAT1 (Phosphoserine aminotransferase 1) is a key enzyme in the phosphorylated pathway of L-serine biosynthesis, which converts glycolytic intermediates into the amino acid serine. Specifically, PSAT1 catalyzes the second step of this three-step pathway - the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to produce O-phosphoserine and alpha-ketoglutarate . This enzymatic activity is critical for generating serine, which serves as a precursor for numerous cellular components including proteins, nucleotides, phospholipids, and other amino acids. PSAT1 is particularly important in rapidly proliferating cells, including cancer cells, where serine metabolism provides essential intermediate metabolites and energy required for maximal growth . The enzyme also contributes to cellular redox balance and one-carbon metabolism, further highlighting its significance in fundamental cellular processes.

What are the key specifications of PSAT1 Antibody, FITC conjugated?

PSAT1 Antibody, FITC conjugated is a polyclonal antibody generated in rabbits using recombinant Human Phosphoserine aminotransferase protein (specifically, amino acids 104-206) as the immunogen . This antibody demonstrates specific reactivity to human PSAT1 and has been validated for ELISA applications . The antibody has undergone Protein G purification with >95% purity and is supplied in liquid form containing a buffer of 50% Glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . The FITC (fluorescein isothiocyanate) conjugation enables direct fluorescent visualization of PSAT1 in experimental settings without requiring secondary antibodies. For optimal performance, the antibody should be stored at -20°C or -80°C upon receipt, with researchers advised to avoid repeated freeze-thaw cycles that could compromise antibody quality .

How should researchers store and handle PSAT1 Antibody, FITC conjugated?

Proper storage and handling of PSAT1 Antibody, FITC conjugated is essential for maintaining its performance and extending its useful life. Upon receipt, the antibody should be stored at -20°C or -80°C according to manufacturer guidelines . FITC conjugation increases the antibody's light sensitivity, so it's crucial to protect the reagent from extended light exposure during storage and handling to prevent photobleaching of the fluorophore. Researchers should aliquot the antibody upon first thaw to minimize freeze-thaw cycles, as repeated freezing and thawing can compromise antibody quality and performance . When working with the antibody, maintain cold chain practices by keeping it on ice or at 4°C during experiment preparation. The antibody is supplied in a buffer containing 50% glycerol, which helps prevent freezing damage, along with 0.01M PBS at pH 7.4 and 0.03% Proclin 300 as a preservative . For optimal results, researchers should follow expiration dates and validate antibody performance if stored for extended periods.

How does PSAT1 post-translational modification affect protein stability and function in cancer cells?

PSAT1 undergoes complex post-translational regulation through acetylation and ubiquitination, which critically determines its stability and function in cancer cells. Recent research has revealed that acetylation serves as a molecular switch regulating PSAT1 degradation, particularly in lung adenocarcinoma (LUAD) . When PSAT1 is deacetylated at lysine 51 (Lys51) by histone deacetylase 7 (HDAC7), this modification enhances the interaction between PSAT1 and the deubiquitinase ubiquitin-specific processing protease 14 (USP14) . This interaction leads to deubiquitination of PSAT1, ultimately resulting in its stabilization and increased protein levels. Conversely, acetylation of PSAT1 promotes its interaction with the E3 ligase ubiquitination factor E4B (UBE4B), leading to increased ubiquitination and subsequent proteasomal degradation . This acetylation-dependent regulation directly impacts serine metabolism and tumor proliferation in LUAD models, with increased PSAT1 stability correlating with enhanced cancer cell growth. These findings suggest that targeting the enzymes controlling PSAT1 acetylation could represent a novel therapeutic strategy for cancers dependent on elevated serine biosynthesis.

What are the critical considerations for optimizing immunofluorescence experiments using FITC-conjugated PSAT1 antibody?

Optimizing immunofluorescence experiments with FITC-conjugated PSAT1 antibody requires careful attention to several experimental parameters. First, researchers must determine the appropriate antibody concentration through titration experiments, typically starting with the manufacturer's recommended dilution (often 1:50 to 1:200) and adjusting as needed based on signal-to-noise ratio. PSAT1 is primarily localized in the cytoplasm, so permeabilization protocols must effectively expose intracellular epitopes without disrupting cellular architecture. Triton X-100 (0.1-0.5%) or saponin (0.1-0.3%) are commonly used permeabilization agents that preserve PSAT1 immunoreactivity. Fixation method also significantly impacts results—4% paraformaldehyde preserves FITC fluorescence better than methanol fixation, which can diminish FITC signal intensity. When designing co-localization studies, researchers should account for FITC's spectral profile (excitation ~495nm, emission ~519nm) to avoid bleed-through with other fluorophores. Additionally, appropriate blocking buffers (typically 5-10% serum from a species different from the antibody host) minimize non-specific binding. For quantitative analyses, researchers should include calibration controls and maintain consistent exposure settings across experimental conditions to enable accurate comparisons of PSAT1 expression levels or localization patterns.

How can researchers validate PSAT1 antibody specificity in their experimental system?

Validating PSAT1 antibody specificity is crucial for ensuring reliable experimental results and should incorporate multiple complementary approaches. First, researchers should perform a positive control experiment using cells or tissues known to express PSAT1, such as lung adenocarcinoma cell lines A549 or H1299, which demonstrate high PSAT1 expression . Comparative analysis with a negative control (cells with PSAT1 knockdown using siRNA or CRISPR-Cas9) provides critical evidence of antibody specificity. Western blot analysis should reveal a single band at the expected molecular weight of PSAT1 (~40 kDa), while multiple bands may indicate cross-reactivity or degradation products. For immunofluorescence applications, pre-absorption controls (pre-incubating the antibody with purified recombinant PSAT1 protein) should substantially reduce or eliminate specific staining patterns. Additionally, comparing staining patterns with multiple PSAT1 antibodies targeting different epitopes can further validate specificity. Researchers should also consider species cross-reactivity limitations, as the FITC-conjugated PSAT1 antibody is specifically validated for human samples . Gene expression correlation studies, comparing antibody signal intensity with known PSAT1 mRNA levels across different cell types, provide another layer of validation. Finally, mass spectrometry analysis of immunoprecipitated proteins can definitively identify antibody-captured proteins.

What experimental approaches can distinguish between PSAT1 expression changes and enzymatic activity alterations?

Distinguishing between changes in PSAT1 expression levels and alterations in its enzymatic activity requires integrated experimental approaches. While immunodetection methods using FITC-conjugated PSAT1 antibody can quantify protein expression, these techniques do not directly measure enzymatic activity. To assess PSAT1 activity, researchers should employ enzyme activity assays that monitor the conversion of 3-phosphohydroxypyruvate to O-phosphoserine, typically by measuring glutamate consumption or α-ketoglutarate production spectrophotometrically. Another approach involves tracing experiments with isotopically labeled substrates (such as 13C-labeled glutamate) followed by mass spectrometry analysis to quantify labeled metabolic products. Researchers should also consider post-translational modifications that affect PSAT1 activity but not necessarily protein levels. For instance, acetylation at Lys51 has been shown to influence PSAT1 stability and potentially its activity . Site-directed mutagenesis experiments creating acetylation-mimicking (K51Q) or acetylation-deficient (K51R) PSAT1 variants can help distinguish between expression and activity effects. Additionally, measuring downstream metabolites in the serine synthesis pathway (serine, glycine) provides functional readouts of PSAT1 activity. For comprehensive assessment, researchers should correlate antibody-based expression measurements with activity assays across multiple experimental conditions to determine whether observed phenotypes result from expression changes or altered specific activity of the enzyme.

What controls should be included when using PSAT1 Antibody, FITC conjugated in cancer research?

When employing PSAT1 Antibody, FITC conjugated in cancer research, inclusion of appropriate controls is essential for result interpretation and validation. Researchers should implement the following control strategy:

For quantitative studies, researchers should additionally include a standard curve using recombinant PSAT1 protein at known concentrations. When studying PSAT1 in relation to serine metabolism, parallel measurements of serine synthesis pathway enzymes (PHGDH, PSPH) provide context for interpreting PSAT1-specific findings. These comprehensive controls enable robust data interpretation and troubleshooting of experimental issues.

How can researchers investigate the relationship between PSAT1 and its regulatory partners?

Investigating interactions between PSAT1 and its regulatory partners requires a multi-faceted experimental approach. Co-immunoprecipitation (Co-IP) experiments represent a powerful starting point, using the PSAT1 antibody to pull down protein complexes followed by immunoblotting for suspected interaction partners like HDAC7 or USP14 . Researchers should consider performing reciprocal Co-IPs (immunoprecipitating the partner and blotting for PSAT1) to further validate interactions. For high-confidence protein interaction mapping, proximity labeling techniques such as BioID or APEX2 can identify proteins within the PSAT1 microenvironment under physiological conditions.

Visualization of protein co-localization can be achieved through dual immunofluorescence microscopy using FITC-conjugated PSAT1 antibody paired with differently labeled antibodies against putative interaction partners. When investigating dynamic interactions, Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) provide real-time assessment of protein-protein proximity in living cells. Functional validation of identified interactions should employ targeted approaches such as siRNA-mediated knockdown or overexpression of regulatory partners to assess consequent effects on PSAT1 stability, localization, and activity .

For studying post-translational modifications like acetylation and ubiquitination that regulate PSAT1, researchers should combine specific detection methods (acetylation-specific antibodies) with modulation of regulatory enzymes (HDAC7, USP14) to establish causal relationships . Molecular techniques including site-directed mutagenesis of key residues (e.g., Lys51 for acetylation) help determine the functional significance of specific modifications .

What approaches can resolve contradictory PSAT1 antibody staining patterns in tissue microarrays?

Resolving contradictory PSAT1 antibody staining patterns in tissue microarrays (TMAs) requires systematic troubleshooting and validation strategies. First, researchers should verify antibody lot consistency, as manufacturing variations may contribute to discrepancies. Comprehensive antigen retrieval optimization is critical—testing multiple methods (heat-induced vs. enzymatic) and buffer conditions (citrate, EDTA, Tris at varying pH) often resolves apparent contradictions. Comparing results from antibodies targeting different PSAT1 epitopes helps distinguish genuine signals from artifacts, with concordant patterns across multiple antibodies suggesting authentic detection.

Researchers should investigate whether post-translational modifications affect epitope accessibility, particularly given that PSAT1 acetylation status varies across tissue types and pathological conditions . When contradictions persist, orthogonal validation using in situ hybridization for PSAT1 mRNA provides expression confirmation independent of protein detection issues. For quantitative assessment, digital image analysis with standardized algorithms reduces subjective interpretation variability. Additionally, researchers should correlate TMA staining patterns with functional assays measuring serine pathway metabolites in matched samples.

The following methodological modifications often improve consistency:

• Titrating antibody concentration specifically for TMA applications

• Extending incubation times to enhance signal uniformity across diverse tissue cores

• Implementing blocking steps to minimize background from endogenous biotin or peroxidase

• Standardizing tissue processing protocols to minimize fixation-related variability

• Incorporating automated staining platforms to enhance reproducibility

How should researchers optimize PSAT1 detection protocols for challenging sample types?

Optimizing PSAT1 detection in challenging samples requires tailored methodological adjustments based on specific sample characteristics. For formalin-fixed paraffin-embedded (FFPE) tissues with potential epitope masking, researchers should implement sequential antigen retrieval protocols, beginning with standard heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) followed by trypsin-based enzymatic digestion if necessary. In tissues with high background autofluorescence (particularly problematic for FITC detection), researchers can employ Sudan Black B (0.1-0.3%) treatment post-immunostaining to quench lipofuscin-derived signals, or alternatively use spectral unmixing during image acquisition.

For frozen tissue sections, optimization of fixation time is critical—typically limiting paraformaldehyde fixation to 10-15 minutes preserves PSAT1 immunoreactivity while maintaining cellular architecture. When working with clinical samples of variable quality, implementing a dual detection strategy combines FITC-conjugated PSAT1 antibody with a secondary detection method (e.g., HRP-based) to compensate for potential fluorescence signal degradation in suboptimal samples.

In metabolically active tissues with high protease content, incorporating additional protease inhibitors (beyond standard cocktails) into sample preparation protocols helps preserve PSAT1 for detection. For samples with abundant extracellular matrix or adipose content, extended permeabilization with detergent mixtures (0.2% Triton X-100 plus 0.05% saponin) improves antibody penetration. Researchers working with samples that demonstrate batch-to-batch variability should implement standardized positive controls within each experimental set and consider normalization to housekeeping proteins of similar abundance and subcellular localization as PSAT1 to facilitate meaningful cross-sample comparisons.