PPIAL4A Antibody

What is PPIAL4A and what is its primary function in cells?

PPIAL4A (Peptidylprolyl Isomerase A-Like 4A), also known as COAS2 (Chromosome 1-amplified sequence 2), is a protein that functions as a peptidyl-prolyl cis-trans isomerase (PPIase). Its primary function is to accelerate protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . This protein is primarily localized in the cytoplasm and has a molecular weight of approximately 18.2 kDa . The gene is located on chromosome 1 in humans, and the protein plays an important role in the proper folding of various intracellular proteins.

What are the common applications for PPIAL4A antibodies in research?

PPIAL4A antibodies are primarily utilized in the following research applications:

These antibodies are predominantly used to detect human PPIAL4A in research contexts, though some may cross-react with other species based on sequence conservation .

What is the difference between monoclonal and polyclonal PPIAL4A antibodies?

Most commercially available PPIAL4A antibodies are polyclonal antibodies, typically generated in rabbits . The distinction between polyclonal and monoclonal antibodies for PPIAL4A research lies in their specificity and application:

• Polyclonal PPIAL4A antibodies: Generated by immunizing rabbits with synthetic peptides, usually corresponding to amino acids 66-92 from the central region of human PPIAL4A . These recognize multiple epitopes on the target protein, potentially providing stronger signals but potentially increased background.

• Monoclonal PPIAL4A antibodies: Less commonly available for PPIAL4A. Would recognize a single epitope, potentially offering higher specificity but potentially lower sensitivity than polyclonal alternatives.

For most general detection purposes in Western blotting and ELISA, the polyclonal antibodies appear to be the standard choice for PPIAL4A research based on current product offerings .

What are the optimal storage conditions for PPIAL4A antibodies to maintain activity?

The proper storage of PPIAL4A antibodies is critical for maintaining their functionality over time. Based on manufacturer recommendations, the following guidelines should be followed:

• Short-term storage (up to 2 weeks): Store at +4°C

• Long-term storage: Aliquot and store at -20°C or below

• Stability: Typically stable for 12 months at -20°C

• Critical consideration: Avoid repeated freeze-thaw cycles as this significantly reduces antibody activity

Some manufacturers provide these antibodies in storage buffers containing preservatives like sodium azide (typically 0.09% or 0.02%) in PBS, sometimes with added stabilizers like glycerol (50%) . Always check manufacturer-specific recommendations as formulations may vary.

What controls should be included when using PPIAL4A antibodies in Western blotting?

When designing Western blot experiments with PPIAL4A antibodies, the following controls should be incorporated:

• Positive control: HEK-293, HeLa, HepG2, or HCT116 cell lysates, which are known to express PPIAL4A

• Negative control:

  • Primary antibody omission

  • Non-specific IgG from the same host species (rabbit)

  • Tissues or cells known not to express the target protein

• Blocking peptide control: Some manufacturers offer the immunizing peptide (e.g., synthetic peptide corresponding to amino acids 66-92) , which can be used to pre-absorb the antibody to verify specificity

• Loading control: Standard housekeeping proteins like GAPDH, β-actin, or α-tubulin to verify equal loading across lanes

Expected molecular weight for PPIAL4A is approximately 18.2 kDa on Western blots . Deviation from this size might indicate post-translational modifications, isoforms, or potential non-specific binding.

How should you optimize antibody dilution for Western blot applications?

When optimizing PPIAL4A antibodies for Western blotting, a systematic approach is recommended:

• Initial dilution assessment: Most manufacturers recommend starting with a 1:1000 dilution for PPIAL4A antibodies in Western blotting

• Titration protocol:

  • Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Use identical samples across blots

  • Keep all other parameters constant (blocking, washing, secondary antibody)

  • Compare signal-to-noise ratio across dilutions

• Optimization factors:

  • Sample type (cell lysate vs. tissue)

  • Protein loading amount (typically 10-50 μg total protein)

  • Detection method (chemiluminescence vs. fluorescence)

  • Incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

• Common issue resolution: If background is high, increase the dilution and optimize blocking conditions. If signal is weak, decrease dilution or extend exposure time.

How can PPIAL4A antibodies be used to study protein-protein interactions?

PPIAL4A antibodies can be valuable tools for investigating protein-protein interactions through several methodologies:

• Co-immunoprecipitation (Co-IP):

  • PPIAL4A antibodies can be used to pull down PPIAL4A and its binding partners

  • Typically requires 0.5-4.0 μg antibody per mg of protein lysate

  • Interacting proteins can be identified through subsequent Western blotting or mass spectrometry

  • Use crosslinking agents to stabilize transient interactions

• Proximity Ligation Assay (PLA):

  • Combination of PPIAL4A antibodies with antibodies against potential interacting proteins

  • Allows visualization of protein interactions in situ

  • Provides spatial information about interaction sites within cells

• Immunofluorescence co-localization:

  • Use PPIAL4A antibodies in combination with antibodies against potential interacting proteins

  • Analyze co-localization patterns using confocal microscopy

  • Quantify co-localization using Pearson's or Mander's coefficients

Since PPIAL4A functions as a PPIase that accelerates protein folding, these methods could help identify its substrates and regulatory partners involved in protein folding pathways .

What are the challenges in detecting PPIAL4A specificity given its similarity to other cyclophilin family members?

PPIAL4A belongs to the cyclophilin family, which includes several highly homologous proteins that function as peptidyl-prolyl isomerases. This presents specific challenges for antibody specificity:

• Sequence homology issues:

  • PPIAL4A shares significant sequence similarity with other cyclophilin family members like PPIA, PPIAL4B, PPIAL4C

  • Cross-reactivity is a potential concern, especially when using antibodies targeting conserved domains

• Validation strategies:

  • Western blot analysis against recombinant PPIAL4A alongside other cyclophilin family members

  • Use of knockout or knockdown models to confirm specificity

  • Epitope mapping to confirm binding to unique regions of PPIAL4A

  • Pre-absorption tests with immunizing peptides

• Technical approaches to improve specificity:

  • Selection of antibodies raised against less conserved regions

  • Use of monoclonal antibodies targeting unique epitopes when available

  • Confirmation with multiple antibodies targeting different epitopes

  • Cross-validation with non-antibody based methods (e.g., mass spectrometry)

When studying PPIAL4A, researchers should carefully consult the specificity validation data provided by manufacturers to ensure that the antibody can distinguish between PPIAL4A and other cyclophilin family members like PPIA (Cyclophilin A), PPIB (Cyclophilin B), PPID (Cyclophilin D), and PPIL4 .

How can PPIAL4A antibodies be used in studies of protein misfolding disorders?

Given PPIAL4A's role in protein folding as a PPIase, its antibodies can be valuable tools in studying protein misfolding disorders:

• Expression analysis in disease models:

  • Western blotting to quantify PPIAL4A expression levels in tissues from disease models vs. controls

  • Immunohistochemistry to examine spatial distribution changes in affected tissues

  • Flow cytometry to analyze PPIAL4A levels in specific cell populations

• Functional studies:

  • Immunoprecipitation to isolate PPIAL4A complexes and identify altered interactions in disease states

  • ChIP-seq using PPIAL4A antibodies to identify potential gene regulatory roles

  • Proximity labeling techniques (BioID, APEX) combined with PPIAL4A antibodies for protein interaction networks

• Therapeutic development:

  • Screening assays using PPIAL4A antibodies to identify compounds that modulate its activity

  • In vivo studies to track PPIAL4A distribution and activity in response to experimental treatments

  • Development of blocking antibodies if PPIAL4A activity contributes to pathogenesis

• Potential disease associations:

  • PPIAL4A's family member PPIA has been linked to various disorders including viral infections like HIV

  • PPIases have been implicated in neurodegenerative diseases involving protein misfolding

  • Cancer research may benefit from PPIAL4A studies as its alternative name (COAS2 - Chromosome 1-amplified sequence 2) suggests potential oncogenic roles

What are common sources of false positives and false negatives when using PPIAL4A antibodies?

When working with PPIAL4A antibodies, researchers should be aware of several potential sources of false results:

Sources of false positives:

• Cross-reactivity with other cyclophilin family members due to sequence homology

• Non-specific binding to highly abundant proteins, particularly when using polyclonal antibodies

• Secondary antibody binding to endogenous immunoglobulins in tissue samples

• Inadequate blocking leading to high background signal

• Post-translational modifications affecting epitope accessibility

Sources of false negatives:

• Epitope masking due to protein-protein interactions or conformational changes

• Fixation-induced epitope alterations, particularly in immunohistochemistry applications

• Insufficient antigen retrieval in fixed tissues

• Degradation of the target protein during sample preparation

• Low sensitivity of detection systems for low-abundance targets

To minimize these issues, implement rigorous validation using multiple techniques, appropriate controls, and optimized protocols specific to the sample type and application.

How should discrepancies in PPIAL4A antibody results between different applications be interpreted?

When faced with discrepancies in results across different applications using PPIAL4A antibodies, consider the following analytical framework:

• Application-specific differences:

  • Western blotting detects denatured proteins, so conformation-dependent epitopes may be affected

  • Immunofluorescence preserves cellular context but may limit antibody accessibility

  • ELISA may detect native or denatured proteins depending on the protocol

• Systematic evaluation process:

  • Confirm antibody validation data for each specific application

  • Evaluate epitope accessibility in different experimental conditions

  • Consider sample preparation differences affecting protein state

  • Examine secondary detection methods and their sensitivity

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Employ complementary non-antibody techniques (e.g., mass spectrometry)

  • Optimize protocols for each specific application

  • Consider the biological context and expected protein levels

• Interpretation framework:

  • Concordant results across applications strengthen confidence

  • Discordant results may reveal biologically relevant insights about protein states or interactions

  • Document all experimental conditions thoroughly to enable accurate comparisons

How can PPIAL4A antibody specificity be independently validated in research applications?

Independent validation of PPIAL4A antibody specificity is critical for research reliability. Consider these methodological approaches:

• Genetic validation methods:

  • CRISPR/Cas9 knockout of PPIAL4A to create negative control samples

  • siRNA or shRNA knockdown to create samples with reduced PPIAL4A expression

  • Overexpression systems to create positive controls with defined PPIAL4A levels

• Biochemical validation approaches:

  • Pre-absorption with immunizing peptide to confirm specific binding

  • Competition assays with purified recombinant PPIAL4A

  • Mass spectrometry validation of immunoprecipitated proteins

  • Parallel testing with multiple antibodies against different epitopes

• Validation across applications:

  • Confirm concordant results across multiple techniques (WB, ELISA, IF)

  • Use orthogonal methods that don't rely on antibodies

  • Correlate protein detection with mRNA expression data

• Documentation standards:

  • Record complete details of validation methods and results

  • Include representative images of controls in publications

  • Report antibody catalog numbers, lots, and dilutions used

  • Share validation data through repositories when possible

For PPIAL4A specifically, validation is particularly important given its similarity to other cyclophilin family members and potential for cross-reactivity .

How are PPIAL4A antibodies being used in studying post-translational modifications?

The investigation of post-translational modifications (PTMs) of PPIAL4A using specific antibodies represents an emerging research area:

• PTM-specific antibody approaches:

  • Development of modification-specific antibodies (e.g., phospho-PPIAL4A, acetyl-PPIAL4A)

  • Combined use of PPIAL4A antibodies with PTM-specific antibodies

  • Sequential immunoprecipitation to enrich for modified forms

• Analytical techniques:

  • 2D gel electrophoresis followed by Western blotting to separate modified forms

  • Immunoprecipitation followed by mass spectrometry for PTM mapping

  • Phosphatase treatment prior to Western blotting to identify phosphorylated forms

• Functional relevance exploration:

  • Correlation of PTM status with enzymatic activity of PPIAL4A

  • Examination of PTM changes under different cellular conditions

  • Investigation of PTM-dependent protein-protein interactions

• Technical considerations:

  • Some PTMs may mask epitopes recognized by standard PPIAL4A antibodies

  • Sample preparation methods must preserve labile modifications

  • Quantitative approaches require careful standardization

While specific PTM studies on PPIAL4A are currently limited, the methodologies established for other cyclophilin family members provide a framework for future research in this area.

What are the considerations for using PPIAL4A antibodies in multiplexed imaging techniques?

As multiplexed imaging techniques gain popularity in research, several considerations apply when incorporating PPIAL4A antibodies:

• Antibody selection criteria for multiplexing:

  • Host species compatibility with other antibodies in the panel

  • Validated performance in the specific fixation conditions required

  • Optimal working concentration to achieve balanced signal across targets

  • Minimal cross-reactivity with other targets in the multiplex panel

• Technical approaches for PPIAL4A in multiplexed imaging:

  • Sequential immunostaining with antibody stripping between rounds

  • Spectral unmixing to resolve overlapping fluorophores

  • Mass cytometry (CyTOF) using metal-conjugated PPIAL4A antibodies

  • DNA-barcoded antibody methods (e.g., CODEX, Immunoseq)

• Optimization strategies:

  • Titration of antibody concentrations in the context of the full panel

  • Careful selection of fluorophores based on expression level and subcellular localization

  • Inclusion of single-stain controls for accurate compensation/unmixing

  • Testing for antibody-antibody interactions or steric hindrance

• Validation approaches:

  • Comparison of staining patterns in multiplexed vs. single-stain experiments

  • Correlation with orthogonal techniques (e.g., Western blot, flow cytometry)

  • Biological validation using samples with known expression patterns

How can PPIAL4A antibodies contribute to understanding the role of this protein in disease mechanisms?

PPIAL4A antibodies can serve as valuable tools for investigating potential roles of this protein in disease pathogenesis:

• Expression profiling in disease tissues:

  • Immunohistochemistry to examine PPIAL4A levels in patient samples

  • Tissue microarray analysis to screen across multiple disease states

  • Quantitative Western blotting to measure expression changes

  • Flow cytometry to analyze PPIAL4A in specific cell populations from patients

• Functional investigation methods:

  • Immunoprecipitation to identify disease-specific interaction partners

  • ChIP-seq to determine if PPIAL4A has chromatin-association roles

  • Proximity ligation assays to map protein interaction networks in disease contexts

  • Function-blocking antibodies to probe PPIAL4A's role in disease models

• Potential disease associations to explore:

  • Cancer biology: The alternative name COAS2 (Chromosome 1-amplified sequence 2) suggests potential roles in cancer

  • Protein misfolding disorders: Given its PPIase activity and role in protein folding

  • Viral infections: Other cyclophilin family members interact with viral proteins

• Translational research applications:

  • Biomarker development using PPIAL4A antibodies in diagnostics

  • Target validation for therapeutic development

  • Patient stratification based on PPIAL4A expression or modification patterns

While current literature on PPIAL4A in disease is limited, the established roles of related cyclophilin family members in various pathologies suggest potential areas for investigation.