AGP5 Antibody

What is AGPAT5 and what is its primary function in cellular metabolism?

AGPAT5 (1-acylglycerol-3-phosphate O-acyltransferase 5) is an enzyme involved in lipid metabolism with a molecular mass of approximately 47 kDa (also calculated as 42 kDa in some references) . It plays a critical role in phospholipid biosynthesis by converting 1-acyl-sn-glycerol-3-phosphate (lysophosphatidic acid or LPA) into 1,2-diacyl-sn-glycerol-3-phosphate (phosphatidic acid or PA). This conversion occurs through the incorporation of an acyl moiety at the sn-2 position of the glycerol backbone .

AGPAT5 demonstrates substrate specificity, acting on LPA containing saturated or unsaturated fatty acids C15:0-C20:4 at the sn-1 position while using C18:1-CoA as the acyl donor. Additionally, it exhibits activity toward lysophosphatidylethanolamine using oleoyl-CoA (but not arachidonoyl-CoA) and lysophosphatidylinositol using arachidonoyl-CoA (but not oleoyl-CoA). Notably, AGPAT5 does not demonstrate detectable activity toward lysophosphatidylglycerol .

What types of AGPAT5 antibodies are available for research applications?

Based on the provided search results, several types of AGPAT5 antibodies are available for research:

These antibodies have been validated for various research applications including Western blotting (WB), immunohistochemistry on paraffin sections (IHC-P), and immunofluorescence/immunocytochemistry (IF/ICC). Both commercially available options are rabbit polyclonal antibodies that recognize human and mouse AGPAT5 .

How does AGP differ from AGPAT5 and what are its primary research applications?

Alpha-1-acid glycoprotein (AGP, also known as orosomucoid or ORM) is fundamentally different from AGPAT5. While AGPAT5 is an enzyme involved in lipid metabolism, AGP is an acute phase inflammatory protein used as a biomarker for inflammation .

AGP serves as a prognostic tool for:

• Predicting risk in inflammatory conditions

• Monitoring response to therapy

• Adjusting nutritional biomarkers for accurate interpretation

AGP can be quantified in multiple biofluids including serum, urine, and saliva, making it versatile for various research contexts. It is increasingly being explored as a rapidly accessible, noninvasive biomarker that requires minimal sample processing and poses fewer biohazard risks compared to traditional serum-based approaches .

Recent research has developed lateral flow immunoassays (LFIA) for AGP quantification with correlations of 0.97 (P < 0.001) for serum, 0.93 for urine, and 0.97 for saliva when compared to reference methods, demonstrating its utility across different sample types .

What are the optimal protocols for validating AGPAT5 antibody specificity in experimental contexts?

Validating antibody specificity is critical for ensuring experimental reliability. For AGPAT5 antibodies, consider the following comprehensive validation approach:

• Epitope verification: Confirm that the antibody recognizes the intended epitope region. For example, antibody ab236541 targets amino acids 150-350 of human AGPAT5 .

• Cross-reactivity testing: Test the antibody against closely related proteins, particularly other AGPAT family members, to ensure specificity. This is especially important given the functional similarities between AGPAT isoforms.

• Multiple detection methods: Validate specificity using at least two independent techniques such as:

  • Western blotting with appropriate molecular weight verification (expected at ~45-47 kDa)

  • Immunohistochemistry with proper controls

  • Immunoprecipitation followed by mass spectrometry

• Knockout/knockdown controls: If possible, use AGPAT5 knockout or knockdown samples as negative controls to confirm antibody specificity.

• Systematic assessment of binding modes: Recent computational approaches can help identify distinct binding modes associated with specific ligands, allowing for more rigorous specificity assessment. This builds on the biophysics-informed model approach that has been successfully used to predict and generate specific antibody variants .

How can I optimize Western blot protocols specifically for AGPAT5 detection?

For optimal Western blot detection of AGPAT5, consider the following methodological approach:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Load 20-30 μg of total protein per lane

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution around the 42-47 kDa range

  • Include molecular weight markers that clearly demarcate the 40-50 kDa range

• Transfer conditions:

  • Use PVDF membrane (0.45 μm pore size) for optimal protein binding

  • Transfer at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary antibody according to manufacturer's recommendation (typically 1:1000)

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly with TBST (4 x 5 minutes)

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit) for 1 hour at room temperature

• Detection and analysis:

  • Use enhanced chemiluminescence (ECL) for detection

  • Verify the band size against the expected molecular weight (42-47 kDa)

  • Include positive controls (human or mouse samples) as the antibodies have been validated for these species

What are the latest computational approaches for designing antibodies with customized specificity profiles for AGPAT5?

Recent advances in computational biology have revolutionized antibody design, allowing researchers to create antibodies with customized specificity profiles. These approaches are particularly relevant for designing antibodies that can specifically recognize AGPAT5 while avoiding cross-reactivity with related proteins:

• Biophysics-informed modeling: This approach associates each potential ligand with a distinct binding mode, enabling prediction and generation of specific variants beyond those observed experimentally. The model is trained on experimentally selected antibodies and can:

  • Predict outcomes for new ligand combinations

  • Generate antibody variants not present in initial libraries that are specific to given combinations of ligands

• Energy function optimization: Novel antibody sequences with predefined binding profiles can be designed by optimizing energy functions associated with each binding mode:

  • For cross-specific sequences: jointly minimize the energy functions associated with desired ligands

  • For specific sequences: minimize energy functions for desired ligands while maximizing those for undesired ligands

• Experimental validation workflow:

  • Conduct phage display experiments with antibody libraries selected against various combinations of ligands

  • Use high-throughput sequencing to analyze selected antibodies

  • Build computational models based on the sequencing data

  • Design and test novel antibody sequences predicted by the model

This integrated experimental-computational approach has been successfully applied to design antibodies with both specific and cross-specific binding properties and could be adapted for developing highly specific AGPAT5 antibodies .

What methods are available for detecting and quantifying AGP across different sample types?

Researchers have multiple methodological options for detecting and quantifying AGP:

• Lateral Flow Immunoassay (LFIA):

  • A recently developed sandwich immunoassay format for AGP where the ratio of test (T) and control (C) line intensities (T/C) is proportional to AGP concentration

  • Working range: 0.25-6.0 μg/mL AGP in buffer

  • Median interassay CV: 13.2% for AGP in buffer

  • Can be used with mobile-based imaging platforms for point-of-care applications

• Sample preparation protocols:

  • Serum: Dilution (typically 1:1000) in assay buffer

  • Urine: Concentrated by ultrafiltration using 10 kDa molecular weight cutoff and diluted 1:2 in buffer

  • Saliva: Centrifuged to remove debris and diluted 1:2 in buffer

• Performance characteristics across sample types:

  Sample Type	Correlation with Reference Method	Sensitivity	Specificity
  Serum	0.97 (P < 0.001)	86%	100%
  Urine	0.93 (P < 0.001)	-	-
  Saliva	0.97 (P < 0.001)	-	-

• Reference methods:

  • Conventional clinical chemistry analyzer

  • Commercial ELISA

These methods offer researchers flexibility in selecting appropriate approaches based on their specific experimental context, available resources, and sample accessibility.

How can researchers troubleshoot inconsistent results when working with AGPAT5 antibodies?

When facing inconsistent results with AGPAT5 antibodies, consider these methodological troubleshooting strategies:

• Antibody validation concerns:

  • Verify antibody lot consistency through lot-specific validation

  • Consider testing multiple antibodies targeting different epitopes of AGPAT5

  • Implement knockout/knockdown controls to confirm specificity

• Sample preparation issues:

  • Ensure complete protein denaturation for Western blotting

  • Optimize tissue fixation for immunohistochemistry (over-fixation can mask epitopes)

  • Address potential post-translational modifications that might affect epitope recognition

• Technical variables:

  • Standardize protein loading and transfer efficiency

  • Control incubation temperatures and times precisely

  • Monitor blocking effectiveness to reduce background

• Species-specific considerations:

  • Confirm species reactivity—both commercially available antibodies mentioned react with human and mouse samples but may have differential affinity

  • Consider sequence homology when working with species not explicitly validated

• Application-specific optimization:

  • For Western blotting: Adjust antibody concentration, incubation time, and membrane type

  • For IHC: Optimize antigen retrieval methods and detection systems

  • For IF/ICC: Refine fixation and permeabilization protocols

• Binding mode analysis:

  • Consider that multiple binding modes may exist for closely related epitopes

  • Apply computational modeling to identify and disentangle these binding modes

How can AGPAT5 antibodies be used to investigate lipid metabolism disorders?

AGPAT5 antibodies offer valuable tools for investigating lipid metabolism disorders through several methodological approaches:

• Expression analysis in disease states:

  • Western blot analysis of AGPAT5 expression in tissue samples from patients with metabolic disorders compared to healthy controls

  • Immunohistochemical staining to assess tissue-specific alterations in AGPAT5 expression and localization

  • Correlation of expression levels with disease severity or clinical parameters

• Functional studies:

  • Co-immunoprecipitation using AGPAT5 antibodies to identify novel interaction partners in normal versus pathological conditions

  • Analysis of AGPAT5 enzyme activity in conjunction with expression levels to determine if post-translational modifications affect function

  • Investigation of AGPAT5's role in metabolic pathways by identifying changes in substrate utilization in disease models

• Mechanistic investigations:

  • Evaluation of AGPAT5's role in lipid droplet formation using immunofluorescence co-localization studies

  • Assessment of phospholipid composition alterations in cellular models where AGPAT5 is dysregulated

  • Examination of AGPAT5's substrate specificity (such as its activity toward LPA containing C15:0-C20:4 fatty acids) in metabolic disease contexts

• Therapeutic target assessment:

  • Use of AGPAT5 antibodies to monitor changes in expression or activity following pharmacological interventions

  • Evaluation of potential compensatory changes in other AGPAT family members when AGPAT5 is targeted

These approaches leverage the specificity of AGPAT5 antibodies to elucidate fundamental mechanisms underlying lipid metabolism disorders and identify potential therapeutic targets.

What are the emerging applications of AGP as an inflammatory biomarker in clinical research?

AGP is gaining recognition as a versatile inflammatory biomarker with several emerging applications in clinical research:

• Multi-biofluid assessment strategies:

  • Development of integrated approaches using serum, urine, and saliva measurements to provide comprehensive inflammatory profiling

  • Correlation studies between AGP levels across different biofluids to establish relationships and determine optimal sampling strategies

  • Validation of non-invasive sampling (urine, saliva) against serum gold standards

• Point-of-care diagnostics development:

  • Implementation of lateral flow immunoassays with mobile-based platforms for rapid quantification

  • Integration with other inflammatory markers (e.g., C-reactive protein) for improved diagnostic accuracy

  • Application in resource-limited settings where conventional laboratory testing is unavailable

• Nutritional status assessment:

  • Adjustment of nutritional biomarkers based on AGP levels to account for inflammation-induced alterations

  • Development of correction factors for various nutritional markers when AGP indicates inflammatory status

  • Implementation in large-scale country-level surveys conducted by the DHS and recommended by the WHO

• Therapeutic monitoring applications:

  • Use of AGP as a prognostic tool for predicting response to anti-inflammatory therapies

  • Longitudinal monitoring of AGP levels to assess treatment efficacy

  • Establishment of threshold values for clinical decision-making in various disease contexts

• Combined biomarker approaches:

  • Development of multi-marker panels incorporating AGP and other inflammatory proteins

  • Calculation of inflammatory indices based on combined biomarker profiles

  • Enhancement of diagnostic and prognostic accuracy through integrated biomarker analysis

What are the future directions for antibody development in AGPAT5 and AGP research?

The field of antibody development for AGPAT5 and AGP research is poised for significant advances in several key areas:

• Integration of computational design and experimental selection:

  • Further refinement of biophysics-informed models to predict antibody specificity with greater accuracy

  • Development of algorithms that can disentangle multiple binding modes associated with closely related epitopes

  • Expansion of antibody design capabilities to generate variants with precisely customized binding profiles

• Enhanced specificity through epitope mapping:

  • More precise targeting of unique AGPAT5 epitopes to minimize cross-reactivity with other AGPAT family members

  • Development of antibodies that can distinguish between different post-translationally modified forms of AGPAT5

  • Creation of conformation-specific antibodies that recognize AGPAT5 in its active versus inactive states

• Multimodal detection platforms:

  • Development of antibody pairs optimized for multiple detection formats (Western blot, IHC, ELISA, etc.)

  • Creation of multiplex assays that can simultaneously detect AGPAT5 along with interacting proteins

  • Integration with advanced imaging techniques for subcellular localization studies

• Point-of-care diagnostics for AGP:

  • Further refinement of lateral flow immunoassays with improved sensitivity and specificity

  • Development of integrated platforms that can quantify multiple inflammatory markers simultaneously

  • Validation across diverse clinical populations to establish reference ranges and decision thresholds

• Therapeutic applications:

  • Development of antibodies that can modulate AGPAT5 activity for potential therapeutic intervention

  • Creation of antibody-drug conjugates targeting cells with aberrant AGPAT5 expression

  • Exploration of engineered antibodies that can cross the blood-brain barrier for neurological applications

These future directions highlight the convergence of computational biology, protein engineering, and clinical diagnostics in advancing antibody development for both research and therapeutic applications related to AGPAT5 and AGP.