PLS3 Antibody, HRP conjugated

What is PLS3/PLSCR3 and what cellular processes does it regulate?

PLSCR3 (phospholipid scramblase 3) is a calcium-dependent protein that mediates accelerated ATP-independent bidirectional transbilayer migration of phospholipids upon binding calcium ions. This activity results in the loss of phospholipid asymmetry in the plasma membrane. Functionally, PLSCR3 plays central roles in several important biological processes, including the initiation of fibrin clot formation, activation of mast cells, and recognition of apoptotic and injured cells by the reticuloendothelial system. PLSCR3 is also a substrate for Protein kinase C (PKC) delta and becomes phosphorylated during apoptosis, with PKC-delta translocating to mitochondria during this process. Research has demonstrated that overexpression of PLSCR3 in HEK293 cells enhances apoptosis induced by UV-irradiation, indicating its significant role in programmed cell death mechanisms .

What are the optimal storage conditions for PLS3 antibody, HRP conjugated?

PLS3 antibodies conjugated with HRP should be stored at -20°C in an appropriate storage buffer. The recommended buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol. To maintain antibody activity and prevent degradation from repeated freeze-thaw cycles, it is advisable to aliquot the antibody into multiple vials before freezing. This approach preserves the functional integrity of the antibody conjugate over extended periods . When properly stored, these antibodies typically maintain their activity for at least one year, though specific manufacturer recommendations should always be followed.

What applications are PLS3 antibody, HRP conjugated most commonly used for?

PLS3 antibody, HRP conjugated is predominantly used in several key immunological applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The recommended dilution range is 1:500-1000. The HRP conjugate eliminates the need for a secondary antibody, allowing for direct detection in one-step procedures .

• IHC-P (Immunohistochemistry on Paraffin-embedded tissues): Recommended dilution range is 1:200-400. The HRP conjugate enables direct colorimetric detection when used with appropriate substrates like DAB or TMB .

• IHC-F (Immunohistochemistry on Frozen tissues): Recommended dilution range is 1:100-500. The conjugated format provides advantages in terms of reduced background and increased specificity compared to two-step detection systems .

The direct HRP conjugation makes these antibodies particularly valuable when minimizing cross-reactivity is important or when working with samples from closely related species.

How does one optimize the signal-to-noise ratio when using HRP-conjugated antibodies in ELISA?

Optimizing signal-to-noise ratio with HRP-conjugated PLS3 antibodies in ELISA requires careful attention to several methodological aspects:

• Antibody titration: Perform a titration experiment using a range of dilutions (typically 1:500 to 1:1000) to determine the optimal concentration that provides maximum specific signal with minimal background .

• Blocking optimization: Use a high-quality blocking buffer containing 1-5% BSA or other appropriate blocking proteins to reduce non-specific binding.

• Incubation protocols: For optimal results, incubate the HRP-conjugated primary antibody at 37°C for 1 hour, followed by thorough washing (five complete washes with wash buffer) .

• Substrate handling: When using chemiluminescent detection, prepare the working solution by mixing equal parts of Luminol/Enhancer Solution and Stable Peroxide Buffer immediately before use. Measure Relative Light Units (RLU) at 425nm within 1-10 minutes following substrate addition for optimal signal intensity .

• Washing stringency: Inadequate washing is a major source of background. Implement five thorough washes after the antibody incubation step to remove unbound antibody .

How can PLS3 antibody, HRP conjugated be used in multiplexed immunoassays with other phospholipid scramblase family members?

Multiplexed detection of different phospholipid scramblase family members (PLSCR1, PLSCR2, PLSCR3/PLS3, etc.) requires careful antibody selection and experimental design:

• Epitope selection: When using HRP-conjugated PLS3 antibodies alongside antibodies targeting other scramblase family members, select antibodies raised against unique epitopes with minimal sequence homology. The PLSCR3 antibody described is derived from a synthetic peptide corresponding to the region 201-295/295 of human PLSCR3, which can be compared to epitopes of other family members to avoid cross-reactivity .

• Sequential detection: For chromogenic detection, use different enzyme conjugates (HRP, AP, etc.) with substrates producing distinct colored products. For PLSCR3/PLS3, the HRP conjugate can be paired with TMB substrate (blue product) or DAB (brown product).

• Spectral separation: In fluorescence-based multiplexing, replace the HRP conjugate with fluorophores having distinct excitation/emission profiles, though this would require using a different conjugate than the HRP version.

• Control experiments: Always include single-staining controls to verify antibody specificity and absence of cross-reactivity or signal interference between detection systems.

Recent research has explored interactions between different PLSCR family members, such as PLSCR2's role as a STAT3 binding partner , highlighting the value of multiplexed approaches in understanding functional relationships between these proteins.

What are the critical considerations when performing in-house HRP conjugation to PLS3 antibodies?

Researchers performing in-house HRP conjugation to PLS3 antibodies should consider several critical factors to ensure optimal conjugate performance:

• Starting antibody quality: Begin with a highly purified PLS3 antibody (>95% purity) to ensure efficient conjugation. Antibodies purified by Protein A affinity chromatography, as used for commercial preparations, provide a good starting material .

• Buffer compatibility: Ensure the antibody is in a carrier-free buffer without preservatives, sodium azide, or high concentrations of primary amines (e.g., Tris, glycine) that might interfere with conjugation chemistry.

• Conjugation chemistry: Lightning-Link® HRP conjugation kits offer a rapid, reproducible method requiring minimal hands-on time (~30 seconds). The process involves adding a modifier to the antibody, incubating for 3 hours, followed by a 30-minute quencher step .

• Antibody recovery: The Lightning-Link® approach provides 100% antibody recovery without the need for further purification, making it ideal for valuable antibody samples .

• Scalability: Modern conjugation methods can be scaled from 10μg to 100mg of antibody while maintaining conjugation efficiency .

• Quality control: Following conjugation, validate the conjugate using a simple ELISA against recombinant PLSCR3 protein, comparing performance to unconjugated primary plus HRP-secondary approach.

The resulting HRP-conjugated PLS3 antibody can be used immediately in applications like Western blotting, ELISA, and immunohistochemistry without further purification steps .

How does calcium dependency affect PLSCR3/PLS3 detection in experimental systems?

PLSCR3/PLS3 is a calcium-dependent phospholipid scramblase, and this characteristic significantly impacts experimental detection approaches:

• Buffer considerations: When detecting PLSCR3 in functional assays, calcium concentration in experimental buffers becomes critical. PLSCR3 undergoes conformational changes upon binding calcium ions, which can expose or mask epitopes recognized by the antibody .

• Activation state detection: The antibody may detect different pools of PLSCR3 depending on whether calcium is present in the experimental system. Some epitopes may only be accessible in the calcium-bound (active) conformation.

• Chelator effects: EDTA or other calcium chelators in sample preparation buffers may alter PLSCR3 conformation and affect antibody recognition. Therefore, calcium concentration should be standardized across all experimental samples.

• Membrane association: PLSCR3 shuttles between cytoplasmic and membrane-associated pools in a calcium-dependent manner. Subcellular fractionation protocols must account for this distribution pattern when preparing samples for immunodetection .

• Quantitative considerations: When quantifying PLSCR3 levels using HRP-conjugated antibodies, researchers should standardize calcium levels in all samples or specifically note whether measurements represent total, active, or inactive protein pools.

What strategies can overcome cross-reactivity issues when using PLS3 antibody, HRP conjugated in tissues with high endogenous peroxidase activity?

Endogenous peroxidase activity can significantly compromise specificity when using HRP-conjugated PLS3 antibodies, particularly in tissues like liver, kidney, and blood cells. Several strategies can mitigate this issue:

• Peroxidase quenching: Prior to antibody application, treat tissue sections with hydrogen peroxide (0.3-3% H₂O₂) in methanol or PBS for 10-30 minutes to inactivate endogenous peroxidases.

• Alternative detection systems: For tissues with exceptionally high endogenous peroxidase activity, consider conjugating the PLS3 antibody to alternative enzymes like alkaline phosphatase instead of HRP.

• Optimized blocking: Use specialized blocking reagents containing avidin/biotin when appropriate, particularly if using avidin-biotin amplification systems alongside the HRP-conjugated antibody.

• Reduced substrate incubation: Minimize the time tissues are exposed to peroxidase substrate to reduce non-specific signal development. Monitor reaction development microscopically to determine optimal endpoint.

• Negative controls: Always include no-primary-antibody controls on serial sections to differentiate between specific PLS3 staining and background from endogenous peroxidase activity.

• Dual validation approach: Confirm HRP-based detection results using a fluorescence-based detection method that eliminates peroxidase-related background concerns.

These approaches are particularly important when performing IHC-P or IHC-F applications where the recommended dilution ranges for PLS3 antibody, HRP conjugated are 1:200-400 and 1:100-500, respectively .

How can PLS3 antibody, HRP conjugated be utilized to investigate the relationship between PLSCR3 and apoptotic pathways?

PLSCR3/PLS3 plays a significant role in apoptotic pathways, particularly through its interaction with PKC-delta. HRP-conjugated PLS3 antibodies can be used in several experimental approaches to investigate these relationships:

• Temporal profiling: Using the HRP-conjugated antibody in time-course Western blot or ELISA experiments to track PLSCR3 expression and phosphorylation status during apoptosis progression. This approach can reveal how PLSCR3 levels change in response to apoptotic stimuli such as UV irradiation .

• Co-localization studies: Combining the HRP-conjugated PLSCR3 antibody with antibodies against apoptotic markers in immunohistochemistry to visualize subcellular relocalization during cell death. The recommended IHC-P dilution (1:200-400) is appropriate for such studies .

• Functional investigations: Using the antibody to detect PLSCR3 in pull-down assays investigating protein-protein interactions with PKC-delta and other apoptotic pathway components.

• Phosphorylation-specific detection: Developing specialized assays that distinguish between phosphorylated and non-phosphorylated PLSCR3 using the HRP-conjugated antibody in combination with phosphatase treatments.

• Mitochondrial association: Tracking PLSCR3 translocation to mitochondria during apoptosis through subcellular fractionation followed by immunodetection with the HRP-conjugated antibody.

Research has shown that overexpression of PLSCR3 in HEK293 cells enhances apoptosis induced by UV-irradiation, establishing its pro-apoptotic function . Using HRP-conjugated antibodies can help further elucidate the mechanisms behind this phenomenon through these experimental approaches.

What is the optimal protocol for using PLS3 antibody, HRP conjugated in chemiluminescent ELISA?

The optimal protocol for chemiluminescent ELISA using HRP-conjugated PLS3 antibody involves several critical steps:

This protocol takes advantage of the direct HRP conjugation to eliminate the need for secondary antibody incubation, reducing protocol time and potential sources of variation. The chemiluminescent approach offers significantly higher sensitivity compared to colorimetric detection, making it ideal for detecting low-abundance PLSCR3 in complex samples .

How can in-house conjugation kits be used to prepare custom PLS3 antibody, HRP conjugated preparations?

Preparing custom HRP-conjugated PLS3 antibodies using in-house conjugation kits involves a straightforward process that can be completed in less than 4 hours:

• Antibody preparation: Ensure the PLS3 antibody is in a compatible buffer free from sodium azide, carrier proteins, and primary amines. If necessary, dialyze the antibody into a suitable buffer (PBS pH 7.2-7.4) .

• Concentration adjustment: Adjust the antibody concentration to 1-4 mg/ml for optimal conjugation efficiency. Low antibody concentrations may result in poor labeling .

• Conjugation process:

  • Add the modifier reagent to the antibody solution (typically 1μl modifier per 10μl antibody)

  • Incubate the mixture for 3 hours at room temperature

  • Add quencher solution (1μl per 10μl of antibody) and incubate for 30 minutes

• Quality control: Test the conjugate in a simple ELISA against recombinant PLSCR3 protein or known positive samples, comparing performance to unconjugated primary plus HRP-secondary approach.

• Storage: Store the freshly conjugated antibody at -20°C in small aliquots to avoid freeze-thaw cycles. Adding 50% glycerol and 1% BSA to the final solution improves stability during storage .

This method provides 100% antibody recovery with no need for additional purification steps, making it highly efficient for laboratory-scale production. The resulting HRP-conjugated PLS3 antibody can be used immediately in applications like Western blotting, ELISA, and immunohistochemistry .

What are the key differences in protocol when using PLS3 antibody, HRP conjugated versus unconjugated primary with HRP-secondary antibody approaches?

The methodological differences between using directly HRP-conjugated PLS3 antibodies versus unconjugated primary with HRP-secondary approach are substantial and affect experiment design, execution, and interpretation:

How can PLS3 antibody, HRP conjugated be utilized in research exploring PLSCR3's role in disease pathogenesis?

PLSCR3/PLS3 has been implicated in several disease processes, particularly those involving dysregulated apoptosis and membrane dynamics. HRP-conjugated PLS3 antibodies provide valuable tools for investigating these connections:

• Cancer research: The anti-apoptotic effects of PLSCR3 overexpression can be studied in tumor tissues using IHC-P applications with the HRP-conjugated antibody. The recommended dilution of 1:200-400 allows for sensitive detection of expression changes in cancer versus normal tissues .

• Cardiovascular disease: PLSCR3's role in fibrin clot formation makes it relevant to thrombotic disorders. ELISA-based quantification using HRP-conjugated antibodies (dilution 1:500-1000) can measure PLSCR3 levels in patient samples versus controls .

• Autoimmune disorders: The involvement of PLSCR3 in the reticuloendothelial system's recognition of apoptotic cells suggests potential roles in autoimmunity. Immunohistochemical methods can map PLSCR3 distribution in affected tissues.

• Neurodegenerative diseases: Apoptotic dysregulation features prominently in many neurodegenerative conditions. IHC-F applications (dilution 1:100-500) can detect PLSCR3 in brain tissue sections .

• Biomarker development: HRP-conjugated antibodies facilitate high-throughput ELISA screening to evaluate PLSCR3 as a potential biomarker across disease states.

Researchers have already begun exploring PLSCR family members as disease mediators, with studies showing that PLSCR2 acts as a STAT3 binding partner and immunomodulator . Similar approaches could unveil PLSCR3's roles across various pathological conditions.

What experimental approaches can determine the specific binding characteristics of PLS3 antibody, HRP conjugated for novel applications?

Characterizing the binding properties of HRP-conjugated PLS3 antibodies is essential for developing novel applications. Several experimental approaches can provide this information:

• Epitope mapping: Using peptide arrays or deletion mutants to precisely identify the recognition site within the PLSCR3 protein. Understanding that the immunogen range for this antibody is 201-295/295 provides a starting point for more detailed mapping .

• Affinity determination: Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) can quantify binding affinity (KD) and binding kinetics (kon, koff) of the HRP-conjugated antibody to recombinant PLSCR3.

• Cross-reactivity profiling: Testing the antibody against recombinant proteins of all PLSCR family members and orthologues from different species to confirm predicted reactivity with human, mouse, rat, cow, sheep, pig, and horse samples .

• Post-translational modification sensitivity: Determining whether phosphorylation status affects antibody recognition, particularly important given PLSCR3's regulation by PKC-delta phosphorylation during apoptosis .

• Conformational dependency: Assessing whether antibody binding is affected by calcium-induced conformational changes in PLSCR3, which would impact experimental design for functional studies.

• Molecular imaging applications: Evaluating if the HRP-conjugated antibody retains functionality when further modified for in vivo imaging applications.

These characterization steps are particularly valuable when adapting the antibody for applications beyond conventional ELISA and IHC, such as in high-content screening or biosensor development.

What are common causes of high background when using PLS3 antibody, HRP conjugated, and how can they be addressed?

High background is a frequent challenge when working with HRP-conjugated antibodies like those targeting PLS3/PLSCR3. Understanding and addressing potential causes is critical for obtaining reliable results:

Implementing a systematic approach to identify and address these potential issues will significantly improve signal-to-noise ratio when using HRP-conjugated PLS3 antibodies across different applications.

How can researchers verify the specificity of PLS3 antibody, HRP conjugated in experimental systems?

Verifying antibody specificity is critical for ensuring reliable experimental results. For HRP-conjugated PLS3 antibodies, several validation approaches should be considered:

• Positive and negative controls:

  • Positive: Tissues or cell lines with confirmed PLSCR3 expression

  • Negative: PLSCR3 knockout tissues/cells or tissues known not to express PLSCR3

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (from the 201-295/295 region) before application to samples. Specific staining should be blocked by this competition .

• Orthogonal detection methods: Compare results obtained with the HRP-conjugated antibody to those from an alternative detection method or a different PLSCR3 antibody targeting a different epitope.

• Western blot validation: Confirm that the antibody detects a band of the expected molecular weight for PLSCR3 (~35 kDa) in Western blot before using in other applications.

• Signal correlation with expression: Demonstrate that signal intensity correlates with PLSCR3 expression levels in systems where expression is experimentally manipulated (overexpression or knockdown).

• Species cross-reactivity verification: If using the antibody across species, confirm specificity in each species rather than assuming the predicted reactivity with human, mouse, rat, cow, sheep, pig, and horse is universal .

• Isotype control experiments: Use an irrelevant HRP-conjugated rabbit IgG at the same concentration to distinguish specific from non-specific binding.

These validation steps provide crucial evidence of antibody specificity, enhancing confidence in experimental results and facilitating accurate interpretation of data.