LIMS1 Antibody, HRP conjugated

What is LIMS1 and why is it a target for antibody-based research?

LIMS1, also known as PINCH1, is widely expressed in mammalian cells and plays an important role in cytoskeletal organization and cell-cell junctions. It forms part of the IPP (ILK-PINCH-Parvin) complex at focal adhesions. Recent research has identified LIMS1 as being strongly expressed in retinal pigment epithelial (RPE) cells, where it contributes to maintaining the outer blood-retinal barrier (oBRB) . LIMS1 has become a significant target for antibody-based research due to its critical role in maintaining cellular architecture and tissue integrity, particularly in epithelial barriers. Additionally, autoantibodies against LIMS1 have been identified as potential biomarkers for pathologic myopia, suggesting immunological relevance beyond its structural functions .

What is the difference between anti-LIMS1 antibodies and anti-LIMS1 autoantibodies?

Anti-LIMS1 antibodies refer to laboratory-produced antibodies specifically designed to target LIMS1 protein for research applications such as Western blotting, immunohistochemistry, and ELISA. These are critical research tools for investigating LIMS1 expression, localization, and function in various experimental contexts. In contrast, anti-LIMS1 autoantibodies are antibodies produced by an organism's immune system against its own LIMS1 protein. Research has identified these autoantibodies in patients with pathologic myopia, where they predominantly belong to IgG1/IgG2/IgG3 subclasses . These autoantibodies have been shown to disrupt the barrier function of retinal pigment epithelial cells and trigger pro-inflammatory responses, suggesting their role in disease pathogenesis rather than as research tools .

How does HRP conjugation enhance LIMS1 antibody functionality in laboratory applications?

Horseradish Peroxidase (HRP) conjugation significantly enhances LIMS1 antibody functionality by providing a direct enzymatic detection system that eliminates the need for secondary antibodies in many applications. This conjugation offers several methodological advantages in LIMS1 research. First, it enables direct detection through catalyzing colorimetric, chemiluminescent, or fluorescent reactions when appropriate substrates are added, allowing visual detection of LIMS1 protein. Second, HRP amplifies the signal through enzymatic reaction, dramatically improving detection sensitivity for low-abundance LIMS1 protein. Third, elimination of secondary antibodies reduces non-specific binding and background noise in experimental results. Fourth, fewer incubation steps are required in protocols like Western blotting and ELISA, improving time efficiency. Finally, the signal produced is proportional to the amount of bound antibody, facilitating quantitative measurements of LIMS1 expression under different experimental conditions.

In which tissue types is LIMS1 protein most abundantly expressed?

According to immunohistochemical and molecular studies, LIMS1 shows differential expression across tissues, with particularly high expression in specific cell types. Immunofluorescent staining and in situ hybridization have confirmed strong LIMS1 expression in the retinal pigment epithelium (RPE), which forms the outer blood-retinal barrier (oBRB) . Western blotting analysis has confirmed LIMS1 expression in multiple ocular cell lines, including human lens epithelial cells (SRA01/04), human trabecular meshwork cells (HTMC), and human RPE cells (ARPE-19), with the highest expression observed in ARPE-19 cells . This expression pattern aligns with LIMS1's important role in maintaining cytoskeletal organization and cell-cell junctions, which are critical for barrier function in epithelial tissues. The high expression in RPE cells specifically suggests a specialized role in maintaining the blood-retinal barrier.

What are the common applications of LIMS1 antibody, HRP conjugated in research?

LIMS1 antibody, HRP conjugated, serves multiple applications in biomedical research related to cytoskeletal dynamics, cell adhesion, and disease mechanisms. In Western blotting, it provides direct detection of LIMS1 protein in cell and tissue lysates without secondary antibodies, as demonstrated in studies confirming LIMS1 expression in ocular cell lines . For quantitative analysis, Enzyme-linked Immunosorbent Assays (ELISA) using HRP-conjugated antibodies enable precise measurement of LIMS1 in biological samples, similar to techniques used to characterize anti-LIMS1 autoantibodies in pathologic myopia studies . Immunohistochemistry and immunofluorescence applications benefit from the direct visualization of LIMS1 localization in cells and tissues, as employed in studies localizing LIMS1 in the posterior pole of the human eye and in ARPE-19 cells . Additionally, these antibodies facilitate investigation of LIMS1's interactions with other focal adhesion proteins and its role in pathologic conditions such as myopia and potential autoimmune disorders.

How can I optimize Western blotting protocols when using LIMS1 antibody, HRP conjugated?

Optimizing Western blotting with LIMS1 antibody, HRP conjugated, requires systematic adjustment of multiple parameters to achieve optimal signal-to-noise ratio. For protein extraction, RIPA lysis buffer has been successfully used for LIMS1 extraction from ocular cell lines, with addition of protease inhibitors to prevent degradation . Protein loading should be calibrated based on expression levels, with 20 μg of protein per lane having proven effective for detection of LIMS1 in previous studies . Transfer conditions require optimization for complete transfer of LIMS1 to the membrane, with PVDF membranes having demonstrated good results in LIMS1 detection. For blocking, use 3-5% BSA or non-fat dry milk in TBST, optimizing blocking time to minimize background without reducing specific signal.

Antibody dilution should start with the manufacturer's recommended concentration (typically 1:1000 to 1:5000) and be adjusted through a dilution series to identify optimal conditions. Incubation with primary antibody overnight at 4°C often provides the best balance between sensitivity and specificity. Thorough washing with TBST between steps is essential for background reduction. For detection, ECL Plus Western Blotting Substrate has been successfully used for visualizing LIMS1 in published research . Include both positive controls (cell lines known to express LIMS1, such as ARPE-19) and loading controls (e.g., β-actin or GAPDH) to validate results and ensure quantitative accuracy.

What are the considerations for validating the specificity of LIMS1 antibody, HRP conjugated?

Validating the specificity of LIMS1 antibody is crucial for generating reliable research data and requires a multi-faceted approach. First, employ positive and negative controls using cell lines or tissues known to express or lack LIMS1. Based on published research, ARPE-19 cells show high LIMS1 expression and serve as an excellent positive control . Second, implement knockout/knockdown validation by comparing signal between wild-type samples and those where LIMS1 has been depleted through CRISPR-Cas9 knockout or siRNA knockdown. Third, conduct peptide competition assays by pre-incubating the antibody with excess purified LIMS1 protein before application to your sample.

Additional validation methods include comparing results using different anti-LIMS1 antibodies targeting distinct epitopes and confirming that detected bands correspond to the expected molecular weight of LIMS1 (approximately 37 kDa). For cross-species applications, test the antibody on samples from different species to assess conservation of the epitope. More rigorous validation can include immunoprecipitation followed by mass spectrometry to confirm that the immunoprecipitated protein is indeed LIMS1. Finally, recombinant expression of tagged LIMS1 in a model system with confirmation of detection using both anti-tag and anti-LIMS1 antibodies provides definitive validation of antibody specificity.

How can LIMS1 antibody, HRP conjugated be used to investigate cytoskeletal organization in RPE cells?

LIMS1 antibody can be strategically employed to investigate cytoskeletal organization in RPE cells through multiple complementary approaches. Immunofluorescence co-staining has been successfully used to examine cytoskeletal organization in ARPE-19 cells by co-staining F-actin and vinculin . LIMS1 antibody can be incorporated into similar protocols to visualize its relationship with these and other cytoskeletal components. Focal adhesion analysis can be performed by using LIMS1 antibody alongside markers for focal adhesions (such as paxillin or FAK) to reveal its role in these structures and their relationship to the cytoskeleton.

For functional studies, RPE cells can be treated with cytoskeleton-disrupting agents (e.g., cytochalasin D, nocodazole) followed by LIMS1 immunostaining to examine changes in its localization and association with remaining cytoskeletal elements. Cell migration assays combined with LIMS1 immunostaining can track its dynamics during RPE cell migration, which involves extensive cytoskeletal remodeling. Barrier function studies are particularly relevant given findings that anti-LIMS1 autoantibodies disrupt barrier function in RPE cells . LIMS1 antibody can monitor expression and localization during barrier formation and disruption, connecting LIMS1 dynamics to functional outcomes.

What are the appropriate controls when using LIMS1 antibody, HRP conjugated in ELISA assays?

Implementing appropriate controls in ELISA assays with LIMS1 antibody, HRP conjugated, is essential for generating reliable and interpretable data. Primary controls should include a coating control using wells coated with known concentrations of purified LIMS1 protein (50 ng of LIMS1 protein has been used effectively in published protocols) . Negative controls should incorporate wells with no antigen (coating buffer only) to assess non-specific binding of the antibody. Isotype controls using an irrelevant HRP-conjugated antibody of the same isotype can evaluate non-specific binding related to the antibody class rather than its specificity.

Technical controls should include blocking controls (wells with blocking buffer but no primary antibody) to assess background from the detection system, and positive controls using samples known to contain LIMS1 (e.g., ARPE-19 cell lysate). Quantitative accuracy requires a standard curve using serial dilutions of purified LIMS1 protein to quantify unknown samples and ensure assay linearity. Specificity can be further validated through pre-absorption controls where LIMS1 antibody is pre-incubated with excess LIMS1 protein to eliminate specific signal. Technical rigor demands multiple wells for each sample to assess assay precision, and inter-assay calibrators (common samples run across multiple plates/days) to normalize between experiments. In published LIMS1 research, the mean absorbance value (A450) of control groups has been used for normalization in quantitative analyses .