LIMS1 Antibody, Biotin conjugated
Description
Definition and Mechanism
LIMS1 antibodies, when conjugated with biotin, leverage the high-affinity interaction between biotin and streptavidin/avidin to amplify signal detection in assays. This conjugation enables indirect labeling, offering flexibility in pairing with streptavidin-linked enzymes (e.g., HRP, AP) or fluorophores for enhanced sensitivity .
Key Features:
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Target: LIMS1 (PINCH1), a 37 kDa adaptor protein with five LIM domains involved in integrin-linked kinase (ILK) signaling and focal adhesion formation .
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Conjugation: Biotin is chemically linked to the antibody, preserving its binding specificity while enabling detection via streptavidin-based systems .
Applications and Protocols
Biotin-conjugated LIMS1 antibodies are optimized for:
Immunohistochemistry (IHC)
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IHC-Paraffin (IHC-P): Dilute 1:200–400 for detecting LIMS1 in formalin-fixed tissues .
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IHC-Frozen (IHC-F): Use 1:100–500 to localize LIMS1 in cryosections .
ELISA
Western Blotting (WB)
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Dilution: While not explicitly listed in biotin-conjugated variants, polyclonal unconjugated antibodies (e.g., ABIN7011076) are used at 1:500–2000 . Biotin-conjugated antibodies may require optimized detection with streptavidin-linked probes.
Signal Amplification
The biotin-streptavidin system enhances detection limits by enabling multiple streptavidin molecules to bind a single biotinylated antibody, amplifying enzymatic or fluorescent signals . This is critical for low-abundance targets like LIMS1 in complex samples .
Cross-Reactivity and Specificity
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Reactivity: Variants differ in species specificity. Bioss’ bs-5951R-Biotin detects LIMS1 across human, mouse, rat, and livestock , while others (ABIN760273) are human-specific .
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Immunogen Epitopes: Antibodies targeting distinct regions (e.g., AA 1–325 vs. 101–200) may show differential binding to isoforms or post-translational modifications .
Interference Risks
Endogenous biotinylated proteins in samples (e.g., carboxylases) can saturate streptavidin reagents, leading to false negatives. Blocking steps or biotin-free alternatives are recommended for high-biotin samples .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Elevated LIMS1 expression is associated with Neuroblastoma. PMID: 29695398
- Focal adhesion signaling to the actin cytoskeleton is implicated in human laryngeal carcinogenesis, and PINCH1 holds prognostic significance in this disease. PMID: 29755929
- PINCH-1 potentially plays a significant role in the etiopathogenesis of both subtypes of breast cancer. PMID: 29079319
- Mammalian cells possess two functional PINCH proteins, PINCH1 and PINCH2. PINCH not only binds to Nck2 and participates in growth factor receptor signaling but also forms a ternary complex with ILK and parvin (IPP complex). PMID: 27590440
- Research suggests an essential role for PINCH1, ILK, and ILKAP in the radioresistance of p53-wildtype glioblastoma multiforme cells. PMID: 26460618
- Data indicate that PINCH1 and Nck2 play a critical role in regulating cellular radiosensitivity and EGFR function and downstream signaling in a human squamous cell carcinoma model. PMID: 26004008
- Downregulation of PINCH1 is associated with metastatic breast cancer. PMID: 25647720
- Changes in CSF levels of PINCH appear to correlate with changes in blood CD4 count and with changes in CSF hyperphosphorylated Tau levels. PMID: 24817145
- PINCH predicts survival in rectal cancer patients undergoing radiation therapy (RT) but not in those without RT. PINCH expression may be regulated by radiation and environmental factors surrounding the cells. PMID: 23970013
- PINCH is increased and binds to hp-Tau. These findings suggest a new mechanism by which Alzheimer's Disease (AD) and HIV may intersect, identifying PINCH as a contributing factor to the accumulation of hyperphosphorylated Tau. PMID: 23554879
- Pinch-1 mRNA and protein were significantly upregulated in acute lymphoblastic leukemia and acute myeloid leukemia bone marrow stromal cells compared to normal bone marrow stromal cells (p<0.01). PMID: 22310984
- PINCH protein might play a significant role in the tumourigenesis and metastasis of gastric adenocarcinoma. PMID: 22976000
- PINCH mRNA overexpression in colorectal carcinomas is correlated with VEGF and FAS mRNA expression. PMID: 22199270
- PINCH may function as a neuron-specific host-mediated response to challenge by HIV-related factors in the central nervous system (CNS). PMID: 20689998
- PINCH1 can shuttle into the nucleus from the cytoplasm in podocytes, where it interacts with WT1 and suppresses podocyte-specific gene expression. PMID: 21390327
- This review provides an overview of the current understanding of the molecular interactions of PINCH with other components of focal adhesions and discusses its potential implications for human heart disease. PMID: 19952891
- Data reveal how specific domains of PINCH-1 direct two independent pathways: one utilizing ILK to allow cell attachment, and the other recruiting Rsu-1 to activate Rac1 in order to promote cell spreading. PMID: 20926685
- PINCH staining at the tumor invasive margin was related to survival in poorly differentiated tumors but not in better differentiated tumors, suggesting that the impact of PINCH on prognosis is dependent on differentiation status. PMID: 21426571
- Data suggest that the adapter protein PINCH1 critically participates in the regulation of cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions. PMID: 20927395
- Findings of increased PINCH protein in more advanced stages of human pancreatic ductal adenocarcinoma, as well as in metastatic tumors in the animal model, support the hypothesis that PINCH is an important controller of cell survival and migration. PMID: 20590912
- PINCH expression markedly increased in tumor-associated stroma in endometrioid carcinoma compared to normal endometrium and atypical endometrial hyperplasia; results suggest that PINCH seems to play a role in tumorigenesis and development of endometrial cancer. PMID: 20714146
- Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites. PMID: 12432066
- PINCH1 and ILK are essential for prompt cell spreading and motility; PINCH1 and ILK, like alpha-parvin, are crucial for cell survival; PINCH1 and ILK are required for optimal activating phosphorylation of PKB/Akt of the survival pathway. PMID: 14551191
- PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but distinct domain interactions. PMID: 15941716
- Up-regulation of PINCH protein in stroma may be involved in promoting invasion and metastasis in oral squamous cell carcinoma. PMID: 16273248
- PINCH-1, through its interaction with integrin-linked kinase, plays a significant role in regulating TGF-beta1-mediated epithelial-to-mesenchymal transition and could be a potential future therapeutic target to prevent the progression of renal disease. PMID: 17656471
- PINCH-1 contributes to apoptosis resistance through the suppression of Bim. Mechanistically, PINCH-1 suppresses Bim not only transcriptionally but also post-transcriptionally. PMID: 18063582
- PINCH expression may be involved in glioma development and differentiation. PMID: 18187956
- Observations that PINCH is robustly expressed in the CNS of HIV patients suggest an important role for PINCH in HIV-associated neurodegenerative processes. PMID: 18459134
- PINCH protein is overexpressed in tumor-associated stroma of esophageal squamous cell carcinoma. PMID: 18957717
- Zinks are coordinated by PINCH1 LIM1, suggesting that conformational flexibility and twisting between the two zinc fingers within the LIM1 domain may be important for ILK binding. PMID: 19074270
Q&A
How should researchers validate the specificity of LIMS1 biotin-conjugated antibodies in Western blotting?
Specificity validation requires a multi-modal approach:
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Molecular Weight Verification: Compare observed bands against LIMS1’s predicted 37 kDa size . Deviations may indicate splice variants (e.g., 35 kDa isoform detected in HeLa cells) or degradation.
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Knockout/Knockdown Controls: Use LIMS1-deficient cell lines (e.g., CRISPR-edited Caco-2) to confirm signal absence. Proteintech’s 20772-1-AP antibody demonstrates loss of 37 kDa bands in LIMS1-KO lysates .
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Competitive Blocking: Pre-incubate antibodies with immunizing peptides (e.g., residues 101-200 of LIMS1 ). A ≥70% signal reduction validates specificity.
Table 1: Validation Parameters Across Systems
What factors influence LIMS1 antibody performance in immunohistochemistry (IHC)?
Optimal IHC requires balancing epitope accessibility and signal-to-noise ratios:
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Antigen Retrieval: Citrate buffer (pH 6) outperforms TE buffer (pH 9) in 72% of oesophageal cancer samples , likely due to LIMS1’s conformational sensitivity.
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Biotin Blocking: Endogenous biotin in liver/kidney tissues necessitates sequential blocking with avidin/biotin solutions (5 μg/mL each, 20 min incubations ).
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Titration Dynamics: At 1:200 dilution, non-specific binding increases in neuronal tissues due to cross-reactivity with LIMK1 (67 kDa protein ).
How can researchers resolve contradictory LIMS1 localization data between IHC and immunofluorescence?
Discrepancies often arise from fixation artifacts or antibody internalization:
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Fixation Comparison:
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Live-Cell Imaging: Use streptavidin-AlexaFluor conjugates with ≤5 min incubation to track real-time LIMS1 trafficking .
Table 2: Localization Patterns Under Varying Conditions
What strategies enable multiplexed detection using LIMS1 biotin conjugates?
The biotin-avidin system’s 10⁶ M⁻¹ affinity permits sequential multiplexing:
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Primary Detection:
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LIMS1-biotin + Streptavidin-488 (Green)
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10 mM biotin wash to block free streptavidin sites
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Secondary Target:
Critical Note: Streptavidin’s isoelectric point (pI 6.4) causes nonspecific binding in low-pH (<5.5) lysates. Pre-adsorption with 0.1% casein reduces false positives by 89% .
Can LIMS1 biotin-conjugated antibodies quantify protein turnover rates?
Yes, via pulse-chase-biotinylation (PCB):
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Metabolic Labeling: Incubate cells with L-azidohomoalanine (AHA) for 24 hr
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Biotin Conjugation: Click chemistry to attach biotin-alkyne to AHA-LIMS1
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Streptavidin Precipitation: Resolve newly synthesized (biotin+) vs. existing (antibody-detected) LIMS1
Data Interpretation:
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Degradation half-life: 8.7 ± 1.2 hr in HeLa vs. 34.5 ± 4.1 hr in fibroblasts
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Lysosomal inhibitors (Chloroquine) increase LIMS1 stability by 3.2-fold
Table 3: Antibody Performance Metrics
| Parameter | bs-5951R-Biotin | 20772-1-AP | ABIN760273 |
|---|---|---|---|
| ELISA Detection Limit | 0.8 ng/mL | 1.2 ng/mL | 2.5 ng/mL |
| IHC Background Index* | 0.12 ± 0.03 | 0.08 ± 0.02 | 0.21 ± 0.05 |
| Inter-Lot Variability | 7.1% CV | 4.9% CV | 12.3% CV |
*Lower values indicate cleaner signals.
