PSAT1 Antibody, HRP conjugated

What is PSAT1 and what cellular functions does it perform?

PSAT1 (Phosphoserine aminotransferase 1) is a member of the class-V pyridoxal-phosphate-dependent aminotransferase family and SerC subfamily. It catalyzes the reversible conversion of 3-phosphohydroxypyruvate to phosphoserine and of 3-hydroxy-2-oxo-4-phosphonooxybutanoate to phosphohydroxythreonine . This enzyme plays a key role in serine biosynthesis pathways. The human PSAT1 gene may also be known by alternative names including PSA1, EPIP, NLS2, PSAT, endometrial progesterone-induced protein, and phosphohydroxythreonine aminotransferase . The protein has a reported molecular weight of approximately 40.4 kilodaltons .

What applications can HRP-conjugated PSAT1 antibodies be used for?

HRP-conjugated PSAT1 antibodies are primarily optimized for ELISA applications, though some variants may support additional techniques. Based on product documentation, specific applications include:

When selecting an HRP-conjugated PSAT1 antibody, researchers should verify the specific clone has been validated for their intended application, as conjugation may affect performance in certain assays compared to unconjugated variants .

What species reactivity do commercial PSAT1 HRP-conjugated antibodies exhibit?

Commercial PSAT1 antibodies conjugated with HRP show varied species reactivity. The following table summarizes reactivity profiles from available products:

For cross-species applications, researchers should verify sequence homology in the antigenic region. Human PSAT1 shares approximately 90.5% amino acid sequence identity with mouse PSAT1 , which may influence antibody cross-reactivity.

What are the optimal conditions for using PSAT1 HRP-conjugated antibodies in ELISA assays?

For optimal ELISA performance with HRP-conjugated PSAT1 antibodies:

• Coating concentration: Use purified PSAT1 protein or cell/tissue lysates at 1-10 μg/ml in carbonate buffer (pH 9.6).

• Blocking: 5% non-fat milk or 3% BSA in PBS for 1.5-2 hours at room temperature.

• Antibody dilution: Most PSAT1 HRP-conjugated antibodies perform optimally at 1:500-1:2000 dilutions, though this should be empirically determined .

• Incubation conditions: 1-2 hours at room temperature or overnight at 4°C.

• Washing buffer: PBS with 0.05-0.1% Tween-20, minimum 3-4 washes between steps.

• Substrate: TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection.

• Stop solution: 2N H₂SO₄ or 1N HCl.

Note that exact conditions may vary based on specific antibody clone and experimental design. Researchers should validate optimal conditions for their specific experimental system .

How does PSAT1 expression change in pathological conditions, and how can HRP-conjugated antibodies help analyze these changes?

PSAT1 expression has been implicated in several pathological conditions:

• Hepatic I/R injury: PSAT1 expression is significantly downregulated after hepatic ischemia/reperfusion (I/R) injury, particularly at the 6-hour timepoint. Research indicates that upregulating PSAT1 expression may alleviate hepatic I/R injury by modulating apoptosis-related pathways .

• Cancer progression: PSAT1 represents a potential therapeutic target in colorectal cancer (CRC) therapy . HRP-conjugated antibodies enable quantitative analysis of PSAT1 expression levels in patient samples via ELISA or immunohistochemistry.

• Neurological disorders: Altered PSAT1 expression may be associated with schizophrenia spectrum conditions and abnormal serine metabolism .

When analyzing pathological PSAT1 expression using HRP-conjugated antibodies, researchers should employ tissue-matched controls and standardized protein loading to enable accurate quantification. For clinical samples, parallel analysis of established biomarkers can provide context for PSAT1 expression changes .

What are the key experimental considerations when validating PSAT1 antibody specificity?

Rigorous validation of PSAT1 antibody specificity is essential for reliable research outcomes. Key validation approaches include:

• Western blot validation: Confirm single band detection at the expected molecular weight (37-40 kDa). Multiple commercial antibodies show specific detection in diverse tissues and cell lines:

  • HeLa cells, HEK-293 cells, Jurkat cells, K-562 cells

  • NIH/3T3 cells

  • Brain tissue from pig, rabbit, rat, and mouse

• Knockout/knockdown controls: siRNA silencing of PSAT1 expression should eliminate antibody signal. Commercial PSAT1 siRNAs with validated knockout efficiency provide essential negative controls .

• Recombinant protein controls: Use purified recombinant PSAT1 (such as E. coli-derived human PSAT1 positions M1-K363 or Q276-L370) as positive controls .

• Cross-reactivity assessment: Test against related proteins, particularly other class-V pyridoxal-phosphate-dependent aminotransferases, to confirm specificity.

• Immunoprecipitation followed by mass spectrometry: This approach provides definitive validation of antibody specificity by confirming the identity of captured proteins.

How can researchers troubleshoot non-specific binding when using HRP-conjugated PSAT1 antibodies?

When encountering non-specific binding with HRP-conjugated PSAT1 antibodies, consider this methodical troubleshooting approach:

• Optimize blocking conditions:

  • Increase blocking time to 1.5-2 hours at room temperature

  • Test alternative blocking agents (5% non-fat milk, 3-5% BSA, or commercial blocking buffers)

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Adjust antibody concentration:

  • Test serial dilutions (1:500 to 1:5000) to determine optimal signal-to-noise ratio

  • For Western blots, high antibody dilutions (1:5000-1:50000) may be optimal for some PSAT1 antibodies

• Modify washing protocols:

  • Increase washing frequency (5-6 washes)

  • Extend washing duration (5-10 minutes per wash)

  • Add additional detergent (0.1-0.2% Tween-20) to washing buffer

• Sample preparation considerations:

  • Ensure complete protein denaturation for Western blot applications

  • Filter lysates to remove particulates

  • Include protease inhibitors to prevent degradation products

• Substrate development:

  • Reduce substrate incubation time to minimize background development

  • Use fresh substrate solutions

  • Consider chemiluminescent substrates with different sensitivity levels

What role does PSAT1 play in the serine biosynthesis pathway and how does this impact experimental design?

PSAT1 occupies a critical position in the serine biosynthesis pathway, which has significant implications for experimental design when studying metabolism:

• Pathway context: PSAT1 catalyzes the second step in the three-step serine biosynthesis pathway, converting 3-phosphohydroxypyruvate to phosphoserine. This pathway links glycolysis to amino acid production, making PSAT1 a critical metabolic junction point.

• Cofactor requirements: PSAT1 activity depends on pyridoxal-5'-phosphate (vitamin B6) as a cofactor. Experimental designs should account for cofactor availability, particularly in in vitro systems .

• Metabolic flux considerations: When studying PSAT1, researchers should consider:

  • Upstream metabolite availability (particularly 3-phosphoglycerate)

  • Potential feedback inhibition mechanisms

  • Interaction with parallel metabolic pathways

• Experimental implications:

  • Cell culture media composition significantly impacts serine pathway activity

  • Serine-free conditions may upregulate endogenous PSAT1 expression

  • Isotope tracing experiments (using labeled glucose or glutamine) can help quantify flux through the PSAT1-catalyzed reaction

• Disease relevance: PSAT1 activity connects to redox homeostasis and nucleotide synthesis, making it particularly important in proliferative contexts including cancer and tissue regeneration after ischemic injury .

What are the recommended controls when using PSAT1 HRP-conjugated antibodies in immunoassays?

For rigorous experimental design with PSAT1 HRP-conjugated antibodies, the following controls should be included:

• Positive controls:

  • Cell lines with validated PSAT1 expression (HeLa, HEK-293, Jurkat, K-562)

  • Tissue lysates with known PSAT1 expression (brain, liver, pancreas)

  • Recombinant PSAT1 protein (particularly for standard curves in quantitative assays)

• Negative controls:

  • PSAT1 knockout or knockdown samples

  • Isotype control antibodies (matched to primary antibody host species and class)

  • Secondary antibody only controls (for unconjugated primary antibodies)

  • Blocking peptide competition (using the immunogenic peptide to confirm specificity)

• Technical controls:

  • Loading controls for Western blots (GAPDH, β-actin)

  • Standard curve samples for quantitative ELISA

  • Unstained samples for background fluorescence in flow cytometry

Including appropriate controls enables confident interpretation of experimental results and facilitates troubleshooting when unexpected results occur.

How does storage and handling affect PSAT1 HRP-conjugated antibody performance over time?

Proper storage and handling of PSAT1 HRP-conjugated antibodies is critical for maintaining performance. Evidence-based recommendations include:

• Storage temperature:

  • Long-term storage: -20°C for glycerol-containing formulations or -80°C for antibodies without cryoprotectants

  • Working aliquots: 4°C for up to one month

• Formulation considerations:

  • Glycerol content: 50% glycerol provides freeze-thaw protection

  • Buffer components: PBS with preservatives (0.02-0.03% sodium azide or 0.03% Proclin-300)

  • pH maintenance: Most formulations maintain pH 7.3-7.4 for optimal antibody stability

• Aliquoting recommendations:

  • Create single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile techniques when handling to prevent microbial contamination

  • Maintain minimum volume (>20μl) to prevent concentration changes from evaporation

• Stability considerations:

  • HRP enzyme activity may decrease over time, even under optimal storage conditions

  • Light exposure should be minimized, particularly after reconstitution

  • Most commercial PSAT1 HRP-conjugated antibodies remain stable for one year from receipt when properly stored

How can PSAT1 HRP-conjugated antibodies be used to investigate the role of PSAT1 in apoptotic pathways?

PSAT1 has been implicated in regulating apoptotic processes, particularly in the context of ischemia/reperfusion injury. HRP-conjugated PSAT1 antibodies can be employed in several methodologies to investigate these mechanisms:

• Quantitative expression analysis:

  • Research has demonstrated that PSAT1 expression is significantly downregulated after hepatic I/R injury, coinciding with increased apoptotic markers

  • HRP-conjugated antibodies enable precise quantification of PSAT1 levels in tissue lysates via ELISA

• Correlation with apoptotic markers:

  • Studies show that PSAT1 upregulation correlates with decreased expression of apoptotic markers including Cyt-c, cleaved caspase 3, and cleaved caspase 9

  • Multiplex ELISA approaches using HRP-conjugated antibodies can simultaneously quantify PSAT1 and apoptotic markers

• Experimental manipulation:

  • PSAT1 overexpression via adeno-associated virus (AAV) vectors has been shown to alleviate hepatic I/R injury

  • HRP-conjugated PSAT1 antibodies can confirm successful overexpression in experimental models

• Mechanistic investigations:

  • PSAT1 may influence apoptosis through metabolic mechanisms, as inhibition of PSAT1 promotes DNA damage and apoptosis

  • Researchers can use HRP-conjugated antibodies in combination with metabolic profiling to correlate PSAT1 levels with specific metabolic states associated with apoptosis resistance

When designing experiments to investigate PSAT1's role in apoptosis, researchers should include both gain-of-function (overexpression) and loss-of-function (siRNA knockdown) approaches to establish causality rather than mere correlation .

What are the differences between various commercially available PSAT1 HRP-conjugated antibodies?

Commercial PSAT1 HRP-conjugated antibodies differ in several key attributes that may influence experimental outcomes:

When selecting between products, researchers should consider their specific experimental requirements and whether published validation data demonstrates efficacy in similar experimental systems .

How can researchers optimize antigen retrieval when using PSAT1 antibodies for immunohistochemistry?

While HRP-conjugated PSAT1 antibodies are primarily used for ELISA, unconjugated PSAT1 antibodies are frequently employed in immunohistochemistry. Optimizing antigen retrieval is critical for successful staining:

• Buffer selection:

  • EDTA buffer (pH 8.0) has shown robust results with PSAT1 antibodies in multiple tissue types

  • TE buffer (pH 9.0) is recommended as primary option for some PSAT1 antibodies

  • Citrate buffer (pH 6.0) may serve as an alternative retrieval method

• Heat-mediated retrieval methods:

  • Pressure cooker: 3-5 minutes at full pressure

  • Microwave: 10-15 minutes at medium power in appropriate retrieval buffer

  • Water bath: 95-98°C for 20-30 minutes

• Tissue-specific considerations:

  • Liver tissue: EDTA buffer (pH 8.0) has been validated for mouse, rat, and human samples

  • Mammary cancer tissue: EDTA buffer (pH 8.0) has shown effective retrieval

  • Brain tissue: May require extended retrieval times due to tissue density

• Enzymatic retrieval alternatives:

  • Some PSAT1 antibodies may benefit from enzymatic retrieval using proteinase K

  • IHC enzyme antigen retrieval reagents with 15-minute incubation have been validated for certain cell types

• Optimization approach:

  • Test multiple retrieval methods in parallel

  • Include positive control tissues with known PSAT1 expression

  • Validate with both immune-positive and immune-negative tissues

What methodological considerations are important when using PSAT1 HRP-conjugated antibodies to analyze PSAT1 expression in diverse tissue types?

When analyzing PSAT1 expression across different tissue types using HRP-conjugated antibodies, researchers should consider these methodological variations:

• Tissue-specific extraction protocols:

  • Brain tissue: Requires gentler homogenization and higher detergent concentrations

  • Liver tissue: Potential interference from abundant proteins necessitates more stringent washing protocols

  • Cell lines: Different lysis buffers may be optimal for adherent versus suspension cells

• Expression pattern expectations:

  • PSAT1 marker can be used to identify specific immune cell populations including CD4 Memory T Cells and Effector Memory CD45RA CD4 T Cells

  • Baseline expression levels vary significantly between tissues, requiring appropriate positive controls

• Sample preparation adjustments:

  • Protein loading: Optimize based on expected PSAT1 abundance (5-30 μg total protein)

  • Blocking conditions: Adjust blocking agent concentration based on tissue-specific background issues

  • Incubation times: May require extension for tissues with dense extracellular matrix

• Data normalization approaches:

  • Select appropriate housekeeping proteins for normalization (GAPDH validated as loading control)

  • Consider tissue-specific reference ranges when interpreting PSAT1 expression levels

  • For quantitative comparisons between tissues, develop tissue-specific standard curves

• Detection system optimization:

  • Signal amplification: May be necessary for tissues with low PSAT1 expression

  • Substrate selection: TMB provides good sensitivity for most applications, but chemiluminescent substrates may be superior for low-abundance detection

By adapting protocols to tissue-specific requirements, researchers can generate more reliable and comparable data across diverse sample types.

How is PSAT1 implicated in metabolic reprogramming during disease progression?

PSAT1 plays significant roles in metabolic reprogramming associated with various disease states:

• Cancer metabolism:

  • PSAT1 represents a therapeutic target in colorectal cancer, potentially through its role in serine metabolism supporting cancer cell proliferation

  • Quantitative analysis of PSAT1 expression using HRP-conjugated antibodies can help measure metabolic adaptation in tumor samples

• Ischemia/reperfusion injury:

  • PSAT1 expression is significantly downregulated during hepatic I/R injury

  • The protective effect of PSAT1 against I/R injury suggests it maintains metabolic homeostasis under stress conditions

  • Research indicates PSAT1 overexpression reduces apoptotic marker expression (Cyt-c, cleaved caspase 3, cleaved caspase 9) while increasing proliferation marker PCNA

• Neurological disorders:

  • Altered PSAT1 expression and serine metabolism have been associated with schizophrenia spectrum conditions

  • PSAT1 may represent a key metabolic junction point connecting amino acid metabolism to neurological function

• Research applications:

  • Metabolic flux analysis using stable isotope-labeled precursors can reveal how PSAT1 activity redirects carbon flow under different conditions

  • Correlation of PSAT1 expression with other metabolic enzymes using multiplex ELISA can map metabolic network adaptations

  • Integrating PSAT1 expression data with metabolomics profiles can provide comprehensive understanding of metabolic reprogramming mechanisms