LPCAT3 Antibody, Biotin conjugated

What is LPCAT3 and what cellular functions does it regulate?

LPCAT3 (lysophosphatidylcholine acyltransferase 3) is a member of the lysophospholipid acyltransferases (LPLATs) family that plays a crucial role in lipid metabolism and homeostasis. It primarily functions by regulating the abundance of different phosphatidylcholine (PC) species in cellular membranes. LPCAT3 specifically regulates the levels of arachidonic PC species, which are essential for maintaining membrane fluidity and function . The enzyme has a calculated and observed molecular weight of 56 kDa and is encoded by the LPCAT3 gene (NCBI Gene ID: 10162) . Recent research has identified LPCAT3 as a target gene of both PPARδ and LXR transcription factors, indicating its regulation is linked to broader metabolic pathways .

What applications are biotin-conjugated LPCAT3 antibodies suitable for?

Biotin-conjugated LPCAT3 antibodies are particularly versatile research tools suitable for multiple applications including Western Blotting (WB), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA) . The biotin conjugation provides significant advantages for detection sensitivity due to the strong interaction between biotin and streptavidin/avidin systems. This characteristic makes these antibodies especially valuable for experiments requiring signal amplification or when working with samples where LPCAT3 expression levels may be low . The antibodies have been validated with human samples, making them suitable for translational research involving human tissues or cell lines .

What is the recommended storage protocol for biotin-conjugated LPCAT3 antibodies?

Biotin-conjugated LPCAT3 antibodies should be stored at -20°C for optimal stability and performance. Under these conditions, the antibody remains stable for up to one year after shipment . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity and prevents microbial contamination . For small volume antibodies (20μl), many preparations contain 0.1% BSA as a stabilizer. It's important to note that aliquoting is generally unnecessary for -20°C storage of these antibodies, which simplifies laboratory workflows and minimizes freeze-thaw cycles that could potentially compromise antibody quality .

How does the transcriptional regulation of LPCAT3 by PPARδ affect experimental design when using LPCAT3 antibodies?

The identification of LPCAT3 as a novel target gene of PPARδ has significant implications for experimental design. Research has demonstrated that PPARδ directly interacts with the proximal PPRE1 motif of the human LPCAT3 gene promoter, leading to increased transcription . When designing experiments with LPCAT3 antibodies, researchers should consider that PPARδ agonists (such as L165041 and GW0742) can increase LPCAT3 expression levels in a dose-dependent manner . This regulatory mechanism creates an important experimental variable that should be controlled or leveraged depending on the research question. For studies examining LPCAT3 protein levels, it's advisable to document the activation status of the PPARδ pathway in the experimental system, as this could significantly impact LPCAT3 expression independent of other variables being tested .

What are the critical considerations when using biotin-conjugated LPCAT3 antibodies in multiplex immunofluorescence studies?

When employing biotin-conjugated LPCAT3 antibodies in multiplex immunofluorescence studies, several critical factors must be addressed. First, endogenous biotin in tissues can generate false-positive signals, necessitating an effective biotin blocking step using avidin/biotin blocking kits prior to antibody application . Second, since the detection system will rely on streptavidin/avidin conjugated to fluorophores, researchers must carefully plan the fluorescence spectrum to avoid overlap with other fluorophores in the multiplex panel. Third, the sequence of antibody application becomes crucial - biotin-conjugated antibodies should typically be applied in earlier steps of sequential staining protocols to allow complete blocking before subsequent antibodies are introduced . Additionally, given that LPCAT3 antibodies have been validated against specific amino acid regions (such as AA 306-363), researchers should verify that these epitopes remain accessible in fixed tissues or cells when designing multiplex protocols .

How does the amino acid specificity of different LPCAT3 antibodies impact experimental outcomes?

The amino acid specificity of LPCAT3 antibodies significantly impacts experimental outcomes and interpretation. Different commercially available antibodies target distinct regions of the LPCAT3 protein - some target amino acids 122-233, others 306-363, while some target amino acids 160-370 . This specificity has several important implications. First, post-translational modifications or protein-protein interactions that mask specific epitopes may affect antibody binding differentially based on the targeted region. Second, alternatively spliced isoforms of LPCAT3 might be recognized by some antibodies but not others depending on whether the target sequence is preserved in all isoforms . The specific sequence targeted by the biotin-conjugated antibody (ABIN1176548) should be verified to ensure it will detect all relevant forms of LPCAT3 in the experimental system. When comparing results across studies, researchers should carefully consider which antibody was used and the specific epitope it recognizes .

What are the optimal dilution ratios for biotin-conjugated LPCAT3 antibodies across different applications?

The optimal dilution ratios for biotin-conjugated LPCAT3 antibodies vary significantly depending on the specific application. For Western Blotting applications, the recommended dilution range is typically between 1:1000 and 1:6000, with the precise dilution requiring optimization for each experimental system . For Immunohistochemistry (IHC) applications, a more concentrated preparation is generally required, with recommended dilutions ranging from 1:150 to 1:600 . For ELISA applications, dilutions should be determined empirically, but generally start at approximately 1:1000 and may require further optimization. It's important to note that these dilution recommendations are sample-dependent, and preliminary titration experiments should be conducted with positive controls to determine the optimal antibody concentration for specific experimental conditions .

What antigen retrieval methods are recommended when using biotin-conjugated LPCAT3 antibodies for immunohistochemistry?

For optimal results when using biotin-conjugated LPCAT3 antibodies in immunohistochemistry, specific antigen retrieval methods are recommended. The primary suggested method is heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0 . This alkaline pH has been shown to effectively expose LPCAT3 epitopes in formalin-fixed, paraffin-embedded tissues. Alternatively, citrate buffer at pH 6.0 can also be used for antigen retrieval, though potentially with different efficacy depending on tissue type and fixation conditions . The choice between these methods may depend on the specific tissue being analyzed, with human stomach and small intestine tissues having demonstrated positive IHC results using these retrieval techniques . Researchers should conduct comparative studies with both retrieval methods on their specific tissue of interest to determine which provides optimal signal-to-noise ratio and epitope accessibility.

How can researchers validate the specificity of biotin-conjugated LPCAT3 antibodies in their experimental systems?

Validating the specificity of biotin-conjugated LPCAT3 antibodies requires a multi-faceted approach. First, researchers should perform Western blot analysis using positive control samples known to express LPCAT3, such as HepG2 cells or pig stomach tissue, which should reveal a band at the expected molecular weight of 56 kDa . Second, a knockdown/knockout validation approach should be employed where possible, using siRNA or CRISPR-Cas9 systems to reduce or eliminate LPCAT3 expression, with subsequent confirmation by Western blot to demonstrate reduced or absent signals . Third, peptide competition assays can be performed, where pre-incubation of the antibody with the immunizing peptide should block specific binding. Fourth, cross-reactivity should be assessed particularly in multi-species studies, noting that while the antibody has demonstrated reactivity with human and pig samples, its reactivity with mouse samples requires independent validation . Finally, for biotin-conjugated antibodies specifically, additional controls should include streptavidin-only staining to rule out endogenous biotin interference.

What are the common causes of background when using biotin-conjugated LPCAT3 antibodies and how can they be mitigated?

High background is a common challenge when working with biotin-conjugated antibodies including LPCAT3 antibodies. The primary cause is endogenous biotin present in many tissues, particularly those with high metabolic activity. To mitigate this, implement a biotin blocking step using commercial biotin/avidin blocking kits prior to antibody application . Insufficient blocking of non-specific binding sites can also contribute to background; use 3-5% BSA or 5-10% normal serum from the same species as the secondary detection reagent for at least 30-60 minutes at room temperature . Over-fixation of tissues can increase non-specific binding; optimize fixation times and consider post-fixation quenching steps if necessary. For streptavidin-based detection systems, using streptavidin conjugates with minimal non-specific binding characteristics and incorporating additional wash steps with PBS-T (0.05-0.1% Tween-20) can significantly reduce background . Finally, if using DAB as a chromogen in IHC, endogenous peroxidase activity should be quenched with 0.3-3% hydrogen peroxide in methanol for 10-30 minutes prior to antibody application.

How can signal amplification be optimized when working with biotin-conjugated LPCAT3 antibodies in samples with low LPCAT3 expression?

Optimizing signal amplification for biotin-conjugated LPCAT3 antibodies in samples with low target expression requires several strategic approaches. First, employ a tyramide signal amplification (TSA) system, which can increase detection sensitivity by 10-100 fold compared to conventional detection methods . This system utilizes the biotin tag already present on the LPCAT3 antibody as a starting point for amplification. Second, use high-sensitivity streptavidin-HRP conjugates followed by enhanced chemiluminescence (ECL) substrates for Western blotting applications. Third, for immunohistochemistry, extend antibody incubation times to 12-18 hours at 4°C to maximize binding to low-abundance targets while maintaining a favorable signal-to-noise ratio . Fourth, reduce the dilution of primary antibody within the recommended range (closer to 1:150 for IHC or 1:1000 for WB) when working with samples known to have low LPCAT3 expression . Finally, incorporate signal enhancement polymers like poly-HRP systems that can attach multiple reporter molecules to each biotin-streptavidin interaction, significantly increasing detection sensitivity without increasing background.

What strategies can be employed to confirm that observed signals originate specifically from the 56 kDa LPCAT3 protein rather than non-specific binding?

Confirming signal specificity for the 56 kDa LPCAT3 protein requires multiple validation strategies. First, always include molecular weight markers on Western blots and verify that the observed band aligns precisely with the expected 56 kDa size of LPCAT3 . Second, run parallel samples treated with LPCAT3-targeting siRNA or from LPCAT3 knockout models to demonstrate signal reduction or elimination . Third, perform peptide competition assays where the biotin-conjugated antibody is pre-incubated with excess immunizing peptide before application; specific signals should be eliminated while non-specific signals would persist . Fourth, compare signals across multiple LPCAT3 antibodies targeting different epitopes (e.g., AA 122-233 vs. AA 306-363) – true LPCAT3 signals should be consistent across different antibodies . Fifth, when performing IHC or IF, compare the staining pattern with known LPCAT3 expression patterns in tissues such as human stomach and small intestine, which have been validated as positive controls . Finally, for technically challenging samples, consider IP-Western approaches where LPCAT3 is first immunoprecipitated with one antibody and then detected via Western blot with the biotin-conjugated antibody to verify specificity.

How does understanding LPCAT3's role in PPARδ and LXR signaling pathways inform antibody selection for specific research questions?

The dual regulation of LPCAT3 by both PPARδ and LXR signaling pathways has significant implications for antibody selection in specific research contexts. Research has demonstrated that PPRE and LXR elements in the LPCAT3 promoter allow independent activation by both transcription factors, with potential additive effects when both pathways are simultaneously activated . When investigating the metabolic intersection of these pathways, researchers should select antibodies that can reliably detect even subtle changes in LPCAT3 protein levels. The biotin-conjugated antibody provides enhanced sensitivity for detecting such changes through signal amplification systems . For studies specifically examining PPARδ-mediated effects, researchers should consider antibodies targeting epitopes that are not affected by post-translational modifications potentially induced by PPARδ signaling . Additionally, when designing experiments involving PPARδ agonists like L165041 or GW0742, researchers should account for the dose-dependent increases in LPCAT3 expression by carefully titrating antibody dilutions to prevent signal saturation in treated samples .

How might emerging super-resolution microscopy techniques benefit from biotin-conjugated LPCAT3 antibodies?

Emerging super-resolution microscopy techniques offer exciting opportunities when paired with biotin-conjugated LPCAT3 antibodies. The biotin-streptavidin system provides an ideal foundation for techniques like STORM (Stochastic Optical Reconstruction Microscopy) and PALM (Photoactivated Localization Microscopy) due to its exceptional binding affinity and specificity . Researchers can leverage this system by using streptavidin conjugated to photoactivatable fluorophores, enabling precise localization of LPCAT3 at the nanoscale level. This approach allows visualization of LPCAT3 distribution within subcellular compartments at resolutions below the diffraction limit (approximately 20nm rather than 200-300nm with conventional microscopy) . For multi-color super-resolution imaging, the biotin-conjugated LPCAT3 antibody can be paired with other directly-labeled antibodies targeting interaction partners, allowing simultaneous visualization of protein complexes involved in lipid metabolism. The strong and specific binding characteristics of the biotin-streptavidin interaction provide excellent signal-to-noise ratios critical for the mathematical reconstruction algorithms used in super-resolution techniques, potentially revealing previously unobservable details of LPCAT3 localization and dynamics .