PPIAL4G Antibody

What is PPIAL4G and what biological functions does it serve?

PPIAL4G (Peptidyl-prolyl cis-trans isomerase A-like 4G) is a member of the peptidylprolyl isomerase family that accelerates protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. This function is critical for proper protein conformation and subsequent cellular activities. PPIAL4G is primarily localized in the cytoplasm and plays important roles in protein folding and trafficking, as well as in cell signaling pathways . Understanding PPIAL4G's function can provide insights into protein structure mechanics and potential targets for therapeutic interventions in various disease contexts .

What types of PPIAL4G antibodies are available for research applications?

Several types of PPIAL4G antibodies are available for research applications:

• By conjugation status:

  • Unconjugated antibodies (most common)

  • FITC-conjugated antibodies (for fluorescence applications)

  • HRP-conjugated antibodies (for enhanced sensitivity in certain assays)

• By host species:

  • Rabbit polyclonal antibodies (most common across manufacturers)

• By clonality:

  • Predominantly polyclonal antibodies are available

PPIAL4G antibodies from various manufacturers have been validated for applications including Western blot, ELISA, immunohistochemistry, and immunofluorescence, with demonstrated reactivity to human and mouse samples .

What is the molecular weight and structure of the PPIAL4G protein?

PPIAL4G is a relatively small protein with the following characteristics:

• Calculated molecular weight: Approximately 18 kDa

• Full amino acid sequence (partial shown): MGIK VQRP RCFF DIAI NNQP AGRV VFEL FSDV CPKT CENF RCLC TGEK GTGK STQK PLHY KSCL FHRV VKDF MVQG GDFS EGNG RGGE SIYG GFFE DESF...

• UniProt Primary Accession: P0DN37

• Gene ID: 644591

• Secondary UniProt Accessions: A1L431, A2BFH1

The protein functions as a peptidyl-prolyl cis-trans isomerase, which requires specific structural domains to catalyze the isomerization reaction. Understanding this structure is essential for designing targeted experimental approaches and interpreting antibody binding results .

What are the recommended applications for PPIAL4G antibody?

PPIAL4G antibodies have been validated for multiple experimental applications:

• Western Blot (WB): Most manufacturers recommend dilutions between 1:500-1:5000, with optimal ranges typically around 1:500-1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated by multiple manufacturers, though specific dilution recommendations vary by antibody source

• Immunohistochemistry (IHC): Typically recommended at dilutions of 1:20-1:200

• Immunofluorescence/Immunocytochemistry (IF/ICC): Recommended at dilutions of 1:200-1:800, particularly useful for subcellular localization studies

• Immunoprecipitation (IP): Some antibodies have been validated for IP applications, typically using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

When selecting a specific application, researchers should verify that the particular antibody has been validated for that use and optimize dilutions for their specific experimental conditions .

How should PPIAL4G antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody performance:

• Storage temperature: Store at -20°C for long-term storage. Some antibodies may be stored at 4°C for short periods

• Storage buffer: Most PPIAL4G antibodies are supplied in buffered solutions containing:

  • PBS (pH 7.3-7.4)

  • 50% glycerol (as cryoprotectant)

  • Small amounts of preservatives (0.02-0.03% sodium azide or 0.03% Proclin 300)

• Handling recommendations:

  • Avoid repeated freeze/thaw cycles

  • Aliquot antibodies upon receipt to minimize freeze/thaw cycles

  • For FITC-conjugated antibodies, avoid exposure to light

  • Allow frozen antibodies to thaw completely before use

  • Briefly centrifuge vials before opening to collect all material

Most manufacturers indicate that properly stored antibodies remain stable for approximately one year after shipment .

What are the recommended protocols for Western blot analysis using PPIAL4G antibody?

For optimal Western blot results with PPIAL4G antibody:

• Sample preparation:

  • Use established cell lines known to express PPIAL4G (validated cell lines include HeLa, HepG2, HEK-293, and Jurkat cells)

  • Prepare whole cell lysates using standard protocols with protease inhibitors

• Gel electrophoresis and transfer:

  • Use standard SDS-PAGE protocols

  • Transfer proteins to nitrocellulose or PVDF membranes

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST

  • Dilute primary PPIAL4G antibody 1:500-1:2000 in blocking buffer

  • Incubate with primary antibody overnight at 4°C

  • Wash membrane thoroughly with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Wash thoroughly before detection

• Detection:

  • Use ECL or other suitable detection system

  • Expected band size: 18 kDa for PPIAL4G , though some sources report observing bands at around 58-65 kDa for related PPIases

• Positive controls:

  • HeLa cells, HepG2 cells, Jurkat cells, and HEK-293 cells have been validated as positive controls

Researchers should optimize these conditions for their specific experimental setup and consider running pilot studies to determine the optimal antibody concentration .

How can I validate the specificity of PPIAL4G antibody in my experiments?

To ensure antibody specificity:

• Positive controls:

  • Use cell lines known to express PPIAL4G (e.g., HeLa, HepG2, Jurkat cells)

  • Include recombinant PPIAL4G protein as a positive control when available

• Negative controls:

  • Use siRNA knockdown of PPIAL4G (siRNA options are available )

  • Include samples from non-expressing tissues when applicable

  • Include secondary antibody-only controls to check for non-specific binding

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide before application

  • This should abolish specific binding if the antibody is truly specific

• Size verification:

  • Confirm that the detected band runs at the expected molecular weight (approximately 18 kDa for PPIAL4G)

  • Note that post-translational modifications may alter apparent molecular weight

• Multiple detection methods:

  • Validate findings using multiple experimental approaches (e.g., WB, IHC, IF)

  • Use antibodies from different manufacturers or that target different epitopes

What factors might affect experimental results when using PPIAL4G antibody?

Several factors can influence experimental outcomes:

• Antibody quality and specificity:

  • Batch-to-batch variation may occur; some manufacturers report >95% purity

  • Epitope accessibility can vary depending on protein conformation and experimental conditions

• Sample preparation:

  • Protein denaturation conditions in Western blotting can affect epitope recognition

  • Fixation methods in IHC/IF can impact antibody binding and signal intensity

  • Tissue or cell lysis conditions may influence protein extraction efficiency

• Expression levels:

  • PPIAL4G expression varies across tissues and may be regulated by various factors

  • Chemical treatments can affect expression (e.g., ammonium chloride, cadmium dichloride, caffeine have been shown to decrease PPIAL4G expression)

• Technical factors:

  • Antibody concentration must be optimized for each application

  • Incubation time and temperature can affect binding efficiency

  • Blocking reagents might interfere with binding

  • Detection method sensitivity can impact results

• Cross-reactivity:

  • PPIAL4G has homology with other PPIase family members, which might lead to cross-reactivity

  • Verify specificity against closely related proteins when possible

How can I troubleshoot weak or absent signal in Western blots using PPIAL4G antibody?

When facing weak or absent signals:

• Protein expression verification:

  • Confirm PPIAL4G expression in your samples via RT-PCR or using a different antibody

  • Use positive control samples known to express PPIAL4G (e.g., HeLa, HepG2 cells)

• Antibody concentration:

  • Increase antibody concentration (start with manufacturer's recommendations, then optimize)

  • Extended primary antibody incubation (overnight at 4°C) may improve signal

• Protein loading:

  • Increase total protein loaded

  • Verify protein transfer efficiency (with Ponceau S or total protein stains)

• Detection sensitivity:

  • Use more sensitive detection reagents (enhanced ECL or fluorescent secondary antibodies)

  • Increase exposure time for chemiluminescent detection

  • Consider using HRP-conjugated PPIAL4G antibody for direct detection

• Sample preparation:

  • Ensure complete protein denaturation for Western blotting

  • Add protease inhibitors to prevent protein degradation

  • Avoid multiple freeze-thaw cycles of protein samples

• Storage and handling:

  • Verify antibody hasn't deteriorated (use fresh aliquots)

  • Follow storage guidelines (typically -20°C with 50% glycerol)

How can PPIAL4G antibodies be utilized in studying protein-protein interactions and cellular signaling?

PPIAL4G antibodies can be valuable tools for investigating protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use PPIAL4G antibodies to pull down protein complexes

  • Analyze interacting partners by mass spectrometry or Western blotting

  • Some PPIAL4G antibodies have been validated for IP applications

• Proximity ligation assay (PLA):

  • Combine PPIAL4G antibody with antibodies against suspected interacting partners

  • Visualize protein-protein interactions in situ with single-molecule resolution

• Chromatin immunoprecipitation (ChIP):

  • If PPIAL4G plays a role in transcriptional regulation, ChIP can identify associated DNA regions

  • Combine with sequencing (ChIP-seq) for genome-wide analysis

• Subcellular localization studies:

  • Immunofluorescence with PPIAL4G antibodies can reveal dynamic localization patterns

  • Co-staining with organelle markers can identify specific compartments

  • FITC-conjugated PPIAL4G antibodies offer direct visualization options

• Signal transduction analysis:

  • Monitor PPIAL4G localization and interactions following cellular stimulation

  • Investigate PPIase activity in different signaling contexts

  • Study phosphorylation status and other post-translational modifications

What approaches can be used to study PPIAL4G in disease models and potential therapeutic applications?

To investigate PPIAL4G in disease contexts:

• Gene knockdown studies:

  • siRNA targeting PPIAL4G is available for knockdown experiments

  • Analyze phenotypic consequences of PPIAL4G depletion

• Tissue expression analysis:

  • Use PPIAL4G antibodies for IHC to compare expression between normal and diseased tissues

  • The Human Protein Atlas contains data on PPIAL4G tissue expression

• Chemical modulation:

  • Several compounds have been shown to affect PPIAL4G expression:

    • Epigallocatechin 3-gallate decreases expression

    • Ammonium chloride affects expression

    • Cadmium dichloride decreases expression

    • Caffeine decreases expression

    • Trichloroethene increases expression

• Functional studies:

  • Investigate PPIase activity in disease models

  • Study the role of PPIAL4G in protein folding under stress conditions

  • Examine potential roles in immunological processes (given the importance of other cyclophilins in immunity)

• Therapeutic targeting:

  • PPIAL4G antibodies can help validate this protein as a potential therapeutic target

  • Inhibitor screening assays may benefit from PPIAL4G antibodies as detection reagents

  • Mucosal delivery studies might benefit from understanding pIgR-mediated transport mechanisms

How can PPIAL4G antibodies be integrated with other molecular tools for comprehensive protein analysis?

Combining techniques enhances research depth:

• Multi-omics integration:

  • Correlate protein expression (via antibody-based methods) with transcriptomic data

  • Combine with metabolomic analysis to understand functional consequences

  • Integrate with interactome mapping to place PPIAL4G in its functional context

• Advanced microscopy:

  • Super-resolution microscopy with PPIAL4G antibodies can reveal detailed subcellular localization

  • Live-cell imaging using cell-permeable antibody fragments or nanobodies

  • FRET studies to examine protein-protein interactions in real-time

  • FITC-conjugated PPIAL4G antibodies are particularly useful for microscopy

• High-throughput screening:

  • Antibody-based assays for compound screens targeting PPIAL4G function

  • Cell-based phenotypic screens following PPIAL4G modulation

• Single-cell analysis:

  • Combine flow cytometry with PPIAL4G antibodies for heterogeneity assessment

  • Single-cell Western blotting to examine expression variation

• Structural biology integration:

  • Use antibodies to validate structural predictions from AlphaFold or similar tools

  • Epitope mapping to connect antibody recognition sites with functional domains

What are the key technical specifications for commercially available PPIAL4G antibodies?

What cell lines and tissues have been validated for PPIAL4G expression detection?

Note: The observed molecular weight for PPIAL4G is reported as 18 kDa by some sources , while PPIL4 (a related protein) is observed at 58-65 kDa . Researchers should be aware of these differences when interpreting their results.

What gene and protein reference information is available for PPIAL4G?