PQLC3 Antibody

What is PQLC3 and why is it relevant to research?

PQLC3 (also known as SLC66A3) is a protein coding gene located on Chromosome 2p25.1 that belongs to the PQ-loop repeat-containing protein family. It functions as a multi-pass transmembrane protein involved in endoplasmic reticulum processes and has been implicated in dolichol-linked oligosaccharide biosynthesis and autophagy regulation . Research interest in PQLC3 has increased due to potential connections to disease models, including associations with prostate cancer and liver pathologies as evidenced by immunohistochemistry studies. The protein is evolutionarily conserved across species (human, mouse, rat, and other mammals), making it valuable for translational research applications.

What types of PQLC3 antibodies are currently available for research?

Several validated PQLC3 antibodies are available for research applications. These include: 1) Rabbit polyclonal antibodies such as HPA061607 from Atlas Antibodies/Sigma-Aldrich, which are affinity-isolated and provided in buffered aqueous glycerol solution ; 2) Conjugated antibodies, including HRP-conjugated anti-PQLC3 antibody targeting amino acids 118-170 and FITC-conjugated PQLC3 polyclonal antibodies for fluorescence-based applications ; and 3) Antibodies validated for specific techniques including immunofluorescence (0.25-2 μg/mL), immunohistochemistry, and ELISA applications . The immunogen sequence commonly used for these antibodies is "FLRYQCYYGYPPLTYLEYPI," which corresponds to a specific epitope of the human PQLC3 protein .

What is the molecular structure and key domains of PQLC3 relevant to antibody targeting?

The PQLC3 protein contains specific functional domains that are important considerations for antibody design and targeting. The partial recombinant form (118-170AA) contains several key functional regions:

This structure is important because antibodies targeting different domains may yield varying experimental results. The PQ-loop motif is particularly significant as it's characteristic of this protein family and involved in key biological interactions. When designing experiments, researchers should consider which domain their antibody targets, as this will impact protein detection in different cellular compartments or conformational states.

How should I design a flow cytometry experiment using PQLC3 antibodies?

When designing a flow cytometry experiment with PQLC3 antibodies, follow this methodological approach: First, select a flow-validated antibody specific to PQLC3, such as the FITC-conjugated PQLC3 polyclonal antibody which is directly labeled for flow cytometry applications . For optimal experimental design, prepare four essential controls: 1) Unstained cells to establish baseline autofluorescence; 2) Negative control cells not expressing PQLC3 to verify antibody specificity; 3) Isotype control antibody (same class as the primary anti-PQLC3 antibody but with no known specificity) to assess non-specific binding; and 4) Secondary antibody control if using indirect staining methods .

To minimize background and improve signal-to-noise ratio, block cells with 10% normal serum from the same host species as the labeled secondary antibody, but ensure this serum is NOT from the same host species as the primary antibody to avoid non-specific signals . Maintain cell concentration between 10^5 to 10^6 cells to prevent clogging and obtain good resolution, but if your protocol involves multiple washing steps, start with 10^7 cells/tube to account for cell loss . Perform all steps on ice using PBS with 0.1% sodium azide to prevent internalization of membrane antigens. For PQLC3 detection, optimal antibody dilution ranges from 1:100-1:500 for most applications, but titration is recommended to determine the optimal concentration for your specific experimental conditions .

What are the recommended protocols for Western blot analysis using PQLC3 antibodies?

For Western blot analysis of PQLC3, a comprehensive experimental protocol should include: Sample preparation beginning with cell lysis using an appropriate buffer containing protease inhibitors, followed by protein quantification using standardized methods such as Bradford assay. Load 20-50 μg of protein per lane on an SDS-PAGE gel (10-12% is typically suitable for detecting PQLC3 with its molecular weight of approximately 21 kDa). After electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane using standard transfer conditions.

Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature, then incubate with primary anti-PQLC3 antibody at a dilution of 1:500-1:2000 (as recommended for polyclonal antibodies against PQLC3). Incubate overnight at 4°C with gentle agitation. After washing 3-5 times with TBST, incubate with an appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature. Perform final washes and develop using ECL substrate. When analyzing results, expect to detect PQLC3 at approximately 21 kDa (isoform b), though post-translational modifications may affect migration. Include positive controls such as PQLC3 overexpression lysates for proper band identification , and negative controls like unrelated protein lysates to confirm antibody specificity.

What approaches should be used for immunohistochemistry (IHC) studies with PQLC3 antibodies?

For successful immunohistochemistry studies with PQLC3 antibodies, follow this methodological approach: Begin with proper tissue fixation using 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding and sectioning at 4-6 μm thickness. Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with heat treatment, as PQLC3 is a transmembrane protein that may require effective epitope unmasking . Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes, then block non-specific binding with 5-10% normal serum from the same species as the secondary antibody.

Apply primary anti-PQLC3 antibody (such as HPA061607) at the recommended dilution, which typically ranges from 1:20-1:200 for IHC applications. Incubate in a humidified chamber overnight at 4°C or for 1 hour at room temperature. After washing with PBS or TBST, apply an appropriate detection system (such as HRP-polymer or ABC method) according to manufacturer's instructions. Develop with DAB substrate, counterstain with hematoxylin, and mount the slides. Include positive control tissues known to express PQLC3 and negative controls (primary antibody omitted) in each experiment. When interpreting results, evaluate membrane and cytoplasmic staining patterns, as PQLC3 is primarily localized to the endoplasmic reticulum . Document staining intensity and distribution across different cell types and tissue compartments for comprehensive analysis.

How can I validate the specificity of my PQLC3 antibody?

Validating the specificity of PQLC3 antibodies requires a multi-faceted approach. Begin with western blot analysis using cells or tissues with known PQLC3 expression alongside a negative control. A specific anti-PQLC3 antibody should detect a band at approximately 21 kDa (for isoform b). Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide (such as the "FLRYQCYYGYPPLTYLEYPI" sequence used for many PQLC3 antibodies) before application to western blot or IHC; specific binding should be abolished or significantly reduced .

Utilize genetic approaches such as PQLC3 knockdown (using PQLC3-targeting siRNA or shRNA) or overexpression systems and verify corresponding decrease or increase in signal intensity . Cross-validate your findings using multiple antibodies targeting different epitopes of PQLC3 - consistent results across different antibodies strengthen confidence in specificity. Consider immunoprecipitation followed by mass spectrometry to confirm that the antibody captures the intended PQLC3 protein. For comprehensive validation, perform immunostaining in cells with fluorescently-tagged PQLC3 and confirm colocalization. Finally, consult validation data provided by manufacturers, such as those available through the Human Protein Atlas for antibodies like HPA061607, which includes tissue arrays of 44 normal human tissues and protein arrays of 364 human recombinant protein fragments .

What are common pitfalls in PQLC3 antibody experiments and how can they be addressed?

Several common pitfalls can affect PQLC3 antibody experiments. First, non-specific binding may occur due to the transmembrane nature of PQLC3. This can be addressed by optimizing blocking conditions (using 5-10% normal serum matching the host species of the secondary antibody) and implementing more stringent washing steps with detergent-containing buffers . Another pitfall is poor signal detection due to insufficient antigen retrieval, particularly in fixed tissues. For PQLC3, which is a membrane protein, use heat-induced epitope retrieval with citrate or EDTA buffer, or consider using proteolytic digestion methods for particularly challenging samples .

False positive results may arise from cross-reactivity with other PQ-loop family proteins due to sequence homology. To address this, select antibodies raised against unique epitopes of PQLC3 and validate using knockout/knockdown controls . Inconsistent results between different experimental techniques (e.g., Western blot vs. IHC) may occur because the epitope accessibility varies depending on the protein's conformation in different sample preparation methods. Use antibodies validated specifically for your application of interest and consider native versus denatured conditions . Finally, batch-to-batch variability can affect reproducibility. Mitigate this by standardizing protocols, using the same lot number for critical experiments, and including appropriate positive and negative controls with each experiment .

How should I interpret PQLC3 antibody signals in relation to its cellular localization?

Interpreting PQLC3 antibody signals requires understanding its expected cellular localization. PQLC3 is primarily localized to the endoplasmic reticulum and is involved in dolichol-linked oligosaccharide biosynthetic processes . In immunofluorescence or immunohistochemistry experiments, expect to observe predominantly cytoplasmic staining with a reticular pattern characteristic of ER proteins, potentially with some perinuclear enrichment. For accurate interpretation, perform co-localization studies with established ER markers such as calnexin or PDI to confirm the expected distribution pattern.

The subcellular location can be affected by experimental conditions: fixation methods that inadequately preserve membrane structures may result in diffuse cytoplasmic staining rather than the expected reticular pattern. Additionally, overexpression systems may cause protein accumulation in unexpected compartments due to saturation of trafficking machinery. When evaluating signal intensity, consider that PQLC3 expression levels vary across tissues and cell types. According to antibody validation data, some tissues may show stronger expression than others, so appropriate positive control tissues should be included for comparison . If investigating potential roles in autophagy, as suggested by research findings, co-staining with autophagosomal markers like LC3 may reveal additional insights into PQLC3 dynamics during cellular stress conditions.

How can PQLC3 antibodies be used in universal CAR-T cell immunotherapy research?

The application of PQLC3 antibodies in universal CAR-T cell immunotherapy research represents an advanced research direction. Based on relevant immunotherapy methodologies, researchers can adapt the universal Fabrack-CAR approach described in related literature . This would involve engineering a universal chimeric antigen receptor containing a non-tumor targeted binding domain that can be redirected through antibody-based mechanisms. For PQLC3 investigation, researchers would first need to establish whether PQLC3 is expressed on target cancer cells through flow cytometry and immunohistochemistry using validated anti-PQLC3 antibodies .

If PQLC3 shows promising expression patterns in cancer cells versus normal tissues, the next step would be to engineer meditope-enabled anti-PQLC3 antibodies that can interact with universal Fabrack-CAR T cells. The experimental design would include in vitro studies to assess T cell activation, proliferation, and IFNγ production in response to PQLC3-expressing target cells, followed by selective killing assays using mixed cell populations . Testing would require multiple methodological steps: flow cytometry to measure CD107a degranulation, cytokine production assays using techniques like ELISA or intracellular cytokine staining, and cytotoxicity assays at various effector-to-target ratios. For in vivo validation, xenograft mouse models would be established with PQLC3-expressing tumor cells, followed by treatment with the engineered antibody system and Fabrack-CAR T cells, with tumor burden monitored via bioluminescence imaging .

What are the considerations for using PQLC3 antibodies in studies of autophagy regulation?

When investigating PQLC3's role in autophagy regulation, several methodological considerations are essential. First, select antibodies that target epitopes that remain accessible during autophagy processes, as protein conformational changes may occur. For immunofluorescence studies, co-staining with established autophagy markers (LC3, p62/SQSTM1, LAMP1) is necessary to track PQLC3 localization during different stages of autophagy. Design experiments that induce autophagy through starvation media (EBSS), rapamycin treatment, or other autophagy inducers, alongside autophagy inhibitors (bafilomycin A1, chloroquine) to differentiate between autophagy induction and block in autophagic flux.

For biochemical analysis, use western blotting with anti-PQLC3 antibodies to monitor changes in PQLC3 protein levels under various autophagy conditions. Combine this with autophagic flux markers such as LC3-I to LC3-II conversion and p62 degradation. Consider proximity ligation assays using PQLC3 antibodies paired with antibodies against known autophagy proteins to detect potential protein-protein interactions during the autophagy process. For functional studies, combine PQLC3 knockdown/knockout approaches with autophagy assays to determine how PQLC3 depletion affects autophagic processes. Advanced researchers might consider using live-cell imaging with fluorescently-tagged PQLC3 antibody fragments to monitor dynamic changes in PQLC3 localization during autophagy in real-time, though this requires careful antibody fragment preparation to maintain specificity while enabling cell penetration.

How can I use structural and biochemical information about PQLC3 to design epitope-specific antibodies for novel applications?

Designing epitope-specific antibodies for PQLC3 requires detailed understanding of its structure and biochemical properties. Based on available structural data, PQLC3 contains distinct functional domains, including the N-terminal membrane anchoring region (118-140), PQ-loop 1 for ligand interaction (141-155), central loop for structural stability (156-165), and C-terminal region with partial catalytic activity (166-170). For novel antibody development, target epitopes should be selected based on their accessibility, uniqueness to PQLC3 (to avoid cross-reactivity with other PQ-loop proteins), and conservation across species if cross-reactivity is desired.

For membrane-spanning regions, careful consideration of hydrophilic exposed loops is crucial, as these are more accessible to antibodies in native conformations. Computational approaches should be employed to predict surface-exposed regions and epitope antigenicity using algorithms like Bepipred, Emini Surface Accessibility, and Kolaskar & Tongaonkar Antigenicity. For applications requiring conformational epitope recognition, focus on the PQ-loop domains which may play crucial roles in protein-protein interactions. Custom antibody development should include recombinant protein production using appropriate expression systems - while E. coli provides high yield, mammalian or insect cell systems offer more native-like post-translational modifications that may be important for epitope structure.

After immunization and antibody production, implement rigorous validation using techniques like epitope mapping with peptide arrays or hydrogen-deuterium exchange mass spectrometry to confirm epitope specificity. For novel applications such as intrabodies or nanobodies targeting specific PQLC3 conformations, yeast or phage display technologies may offer advantages for selecting high-affinity binders to conformational epitopes.

How do different PQLC3 antibody formats compare in terms of applications and limitations?

Conjugated antibody formats substantially expand application possibilities: HRP-conjugated anti-PQLC3 antibodies are optimized for ELISA and Western blot applications, providing direct detection without secondary antibodies . FITC-conjugated PQLC3 antibodies enable direct visualization in flow cytometry and immunofluorescence, eliminating secondary antibody cross-reactivity concerns . The comparative effectiveness of these formats varies by application:

For emerging super-resolution microscopy techniques, directly labeled antibody fragments (Fab, F(ab')2) may offer advantages by providing reduced steric hindrance and better epitope accessibility in complex cellular structures where PQLC3 resides . When selecting between formats, researchers should consider the detection sensitivity requirements, whether quantitative analysis is needed, and the specific cellular compartments where PQLC3 needs to be visualized.

How can I design multiplexed immunoassays that include PQLC3 detection alongside other biomarkers?

Designing multiplexed immunoassays for PQLC3 alongside other biomarkers requires careful consideration of antibody compatibility, spectral overlap, and optimization strategies. For immunofluorescence or flow cytometry applications, select anti-PQLC3 antibodies conjugated to fluorophores with minimal spectral overlap with other target fluorophores. FITC-conjugated PQLC3 antibodies work well when paired with markers labeled with far-red fluorophores like Cy5 or APC . When designing panels, pair PQLC3 detection with relevant markers based on research context - for endoplasmic reticulum studies, include ER markers like calnexin or PDI; for autophagy studies, include LC3 and p62.

For multiplex immunohistochemistry, sequential immunostaining protocols can be employed using antibodies from different host species to avoid cross-reactivity. This typically involves cycles of primary antibody application, detection, signal development, and antibody stripping before the next cycle . Tyramide signal amplification (TSA) methods allow multiple antibodies from the same host species to be used sequentially by permanently depositing fluorophores and completely removing antibodies between cycles. When developing multiplex Western blot assays, select antibodies with target proteins of sufficiently different molecular weights from PQLC3 (~21 kDa) to avoid signal overlap.

For all multiplex approaches, thorough validation is essential: perform single-marker controls alongside multiplexed samples to ensure antibody performance is not compromised by multiplexing. Test for potential cross-reactivity between antibodies and secondary detection systems. Implement computational approaches for signal unmixing if spectral overlap exists, especially in highly multiplexed imaging approaches. Finally, consider sample preparation carefully, as fixation methods may differentially affect epitope accessibility for PQLC3 versus other biomarkers in your panel.

What are the emerging applications of PQLC3 antibodies in understanding disease mechanisms?

Emerging applications of PQLC3 antibodies in disease mechanism research span several promising areas. In cancer research, PQLC3 has been implicated in prostate cancer and liver pathologies, suggesting potential as a biomarker or therapeutic target. Anti-PQLC3 antibodies can be employed in tissue microarray analysis to evaluate expression patterns across multiple tumor types and correlate with clinical outcomes. The methodology would involve standardized immunohistochemistry protocols with validated anti-PQLC3 antibodies followed by digital pathology quantification for objective assessment of expression levels .

In autophagy-related disorders, including neurodegenerative diseases, PQLC3's role in lysosomal transport of autophagy substrates makes it relevant for studying disease mechanisms. Researchers can utilize high-content imaging approaches with anti-PQLC3 antibodies to track changes in protein localization and abundance in cellular models of disease. This would involve automated microscopy coupled with quantitative image analysis to detect subtle alterations in PQLC3 distribution patterns or colocalization with disease-relevant proteins.

For immunotherapy applications, the use of PQLC3 antibodies in universal CAR-T approaches represents a cutting-edge direction. Based on methodologies described in related research, engineered antibodies against PQLC3 could potentially be used to redirect universal CAR-T cells toward cancer cells expressing PQLC3 . This would involve comprehensive profiling of PQLC3 expression across normal and malignant tissues to identify suitable therapeutic windows, followed by in vitro and in vivo efficacy testing using protocols similar to those described for other targets in universal CAR-T systems.

In infectious disease research, particularly relevant in the context of COVID-19, antibody technologies developed for SARS-CoV-2 research could be adapted to study potential interactions between viral components and host factors like PQLC3. If PQLC3 plays roles in viral entry, replication, or immune evasion mechanisms, anti-PQLC3 antibodies could be valuable tools for mechanistic studies and potential therapeutic development .