CLCA1 Antibody, Biotin conjugated

What is CLCA1 and why is it important in research?

CLCA1 (Chloride Channel Accessory 1) is a secreted protein containing an N-terminal zinc-dependent metallohydrolase domain with a conserved HExxE catalytic motif similar to that found in matrix metalloproteases (MMPs) and ADAMs (A Disintegrin And Metalloproteases). It undergoes intracellular autocatalytic cleavage, resulting in two cleavage products that are both secreted . CLCA1 is particularly significant in research because it activates calcium-dependent chloride currents and has been identified as a key regulator of TMEM16A/Anoctamin1, playing important roles in chronic inflammatory airway diseases such as asthma, COPD, and cystic fibrosis, as well as in certain cancers . Understanding CLCA1 function provides insights into mucus production regulation and airway physiology.

What are the typical applications for CLCA1 antibodies?

CLCA1 antibodies can be utilized across multiple experimental applications. According to the available data, standard CLCA1 antibodies are typically validated for Western Blot (WB), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA) applications . Some antibodies have expanded applications including Immunoprecipitation (IP) and Immunofluorescence (IF) . For Western blotting applications, CLCA1 antibodies are generally recommended at dilutions ranging from 1:500 to 1:2000, while for immunohistochemistry, dilutions between 1:50 and 1:500 are typically suggested . These applications allow researchers to detect, quantify, and localize CLCA1 in various biological samples.

What is the difference between standard and biotin-conjugated CLCA1 antibodies?

Standard CLCA1 antibodies are typically unconjugated, meaning they require a secondary detection system (such as a secondary antibody) for visualization in experimental assays . Biotin-conjugated CLCA1 antibodies have biotin molecules covalently attached, which can directly bind to streptavidin-conjugated detection systems. This biotin-streptavidin interaction offers several advantages including: (1) enhanced sensitivity due to the high affinity between biotin and streptavidin, (2) amplification of signal as multiple streptavidin molecules can bind to each biotin, and (3) compatibility with various detection systems such as streptavidin-HRP, streptavidin-fluorophores, or streptavidin-PE for flow cytometry . Biotinylation does not adversely affect the antibody's function, as demonstrated in whole-cell patch clamp experiments comparing purified N-CLCA1 and purified biotinylated N-CLCA1 .

How is the N-terminal fragment of CLCA1 biotinylated for research purposes?

The N-terminal fragment of CLCA1 (N-CLCA1, residues 22-694) can be biotinylated through a specific methodological protocol. First, the N-CLCA1 sequence is cloned into a pHL-Avitag3 vector, which incorporates a BirA biotin ligase recognition motif and hexahistidine tag at the C-terminus . This construct is then transiently transfected into 293F cells using Hype-5 at a 1:1.5 μg DNA:μl Hype-5 ratio (using 1 μg of plasmid DNA per 1 million cells) . After expressing for 72 hours, the secreted protein is harvested from media supernatant and purified using Ni-NTA chromatography. For in vitro biotinylation, the purified N-CLCA1 is exchanged into a specific buffer (100 mM Tris pH 7.5, 200 mM NaCl, and 5 mM MgCl₂) and specifically biotinylated by adding biotin and Escherichia coli BirA ligase at 4°C overnight . Excess biotin is then removed using a desalting column. This biotinylated N-CLCA1 can be used at concentrations of 10-50 μg/ml for experimental applications .

What are the expected molecular weights when detecting CLCA1 with antibodies?

CLCA1 exhibits differences between calculated and observed molecular weights, which is important to consider when interpreting experimental results. The data shows:

This discrepancy is due to CLCA1's autocatalytic cleavage properties . The full-length CLCA1 protein undergoes self-cleavage resulting in two fragments. When using antibodies targeting different epitopes of CLCA1, researchers might observe different bands corresponding to either the full-length protein or one of its cleaved fragments. The N-terminal fragment, which is often used in biotinylated form for research applications, contains the functional metallohydrolase domain responsible for CLCA1's regulatory activities . Researchers should be aware of these molecular weight variations when designing experiments and interpreting results from Western blots or other size-based detection methods.

What control experiments should be performed when using biotinylated CLCA1 antibodies?

When using biotinylated CLCA1 antibodies or biotinylated N-CLCA1 fragments, several control experiments are essential to ensure data validity. First, include a buffer control where the same volume of buffer (without the biotinylated protein) is added to cells to establish baseline measurements . When conducting binding assays, include a streptavidin-PE alone control to determine background binding levels . For blocking experiments, include both relevant blocking antibodies (such as antibodies against potential interaction partners like TMEM16A) and appropriate isotype controls to distinguish specific from non-specific blocking . Additionally, include control antibodies targeting irrelevant or inaccessible epitopes (like intracellular domains of transmembrane proteins when performing cell-surface binding assays) to confirm binding specificity . Finally, validate that biotinylation does not affect protein function by comparing the activities of non-biotinylated and biotinylated proteins in functional assays, such as whole-cell patch clamp experiments for CLCA1 .

How can biotinylated CLCA1 be used to study CLCA1-TMEM16A interactions?

Biotinylated CLCA1, specifically the N-terminal fragment (N-CLCA1), can be employed in flow cytometry-based binding assays to study interactions with TMEM16A. In this methodology, biotinylated N-CLCA1 is pre-incubated with streptavidin-phycoerythrin (SA-PE) at a 4:1 molar ratio for 15 minutes at room temperature to produce fluorescently labeled tetramers of N-CLCA1 (N-CLCA1/SA-PE) . Cells (approximately 4 × 10⁵ cells/sample) are then stained with either SA-PE alone (as control) or the N-CLCA1/SA-PE complex diluted in PBS containing 1% BSA at 4°C . To validate specific binding to cell surface TMEM16A, blocking experiments can be performed using antibodies targeting extracellular epitopes of TMEM16A, such as goat-anti-human-TMEM16A polyclonal antibody S-20, which targets residues within the last extracellular loop (residues 820-870) . Control antibodies should include isotype controls and antibodies targeting intracellular domains that should not interfere with cell-surface interactions. After staining, cells are washed and analyzed by flow cytometry. This technique has revealed that N-CLCA1 directly engages TMEM16A on the cell surface, providing critical insights into how CLCA1 regulates calcium-dependent chloride channels .

What functional assays can demonstrate the biological activity of biotinylated CLCA1?

The biological activity of biotinylated CLCA1 can be assessed through various functional assays, with whole-cell patch clamp experiments being particularly informative. In these assays, biotinylated N-CLCA1 (typically at 10-50 μg/ml) is applied to untransfected cells for 24 hours prior to electrophysiological measurements . The activation of calcium-dependent chloride currents (ICaCC) can then be measured in the presence of 10 μM intracellular Ca²⁺ and physiological concentrations of extracellular Cl⁻ . Robust, slightly outward rectifying currents indicate functional activity of the biotinylated protein. Importantly, comparative experiments between non-biotinylated and biotinylated N-CLCA1 have demonstrated that both proteins can robustly activate these currents, confirming that biotinylation does not adversely affect function . Additional functional assays include co-culture experiments where cells expressing GFP are co-cultured with cells transfected with CLCA1 or control vectors to assess paracrine activation of chloride currents, and conditioned medium experiments where cells are exposed to medium from CLCA1-expressing cells to demonstrate secreted CLCA1's activity .

How do you optimize immunostaining protocols using biotin-conjugated CLCA1 antibodies?

Optimizing immunostaining protocols with biotin-conjugated CLCA1 antibodies requires careful consideration of several parameters. For immunohistochemistry applications, antigen retrieval methods significantly impact staining quality. The data indicates that for CLCA1 detection in human small intestine tissue, TE buffer pH 9.0 is suggested for antigen retrieval, though citrate buffer pH 6.0 can be used as an alternative . Antibody dilution requires careful titration in each testing system, with recommended ranges for IHC being 1:50-1:500 . For immunofluorescence applications using biotin-conjugated antibodies, blocking endogenous biotin in tissues is crucial to reduce background, which can be accomplished with avidin/biotin blocking kits. When detecting CLCA1 in different sample types, researchers should consider the validated reactivity of their antibody; the available data indicates reactivity with human and mouse samples . Additionally, for multicolor immunofluorescence, careful selection of streptavidin conjugates with compatible fluorophores is essential to avoid spectral overlap with other detection channels.

Why might CLCA1 detection show unexpected banding patterns in Western blots?

Unexpected banding patterns when detecting CLCA1 in Western blots can occur due to several factors related to the protein's biology. The primary reason is CLCA1's autocatalytic cleavage property. CLCA1 undergoes self-cleavage resulting in multiple fragments, which can appear as distinct bands on Western blots . The calculated molecular weight for full-length CLCA1 is approximately 100 kDa (914 amino acids), but the observed molecular weight is often around 50 kDa due to this cleavage process . A previous study suggested that a band around 75 kDa corresponds to the N-terminal autocatalytic fragment . Additionally, differences in glycosylation patterns across different cell types and tissues can alter the apparent molecular weight of CLCA1. Researchers may observe different banding patterns depending on which epitope the antibody recognizes (N-terminal versus C-terminal fragments) and the specific tissue or cell type being examined. When troubleshooting unexpected banding patterns, consider using positive controls like COLO 320 cells, mouse colon tissue, or mouse lung tissue, which have been validated for CLCA1 detection .

How can non-specific binding be minimized when using biotin-conjugated CLCA1 antibodies?

Minimizing non-specific binding when using biotin-conjugated CLCA1 antibodies requires implementing several strategic approaches. First, pre-block samples with human FcR blocking reagent (1:100 dilution for 15 minutes) before applying antibodies, particularly for flow cytometry applications . Include appropriate buffer controls in all experiments, where the same volume of buffer (without the biotinylated protein) is added to cells to establish true baseline readings . For flow cytometry and cellular binding assays, dilute reagents in PBS containing 1% BSA (FACS buffer) to reduce non-specific interactions . When conducting blocking experiments, include not only relevant blocking antibodies but also appropriate isotype controls to distinguish specific from non-specific blocking effects . Additionally, when working with tissues, consider pre-blocking endogenous biotin using avidin/biotin blocking kits, particularly for samples from biotin-rich tissues like liver, kidney, or brain. Finally, optimize antibody concentrations through careful titration, as concentrations that are too high can lead to increased background and non-specific binding. The recommended dilution ranges for standard CLCA1 antibodies are 1:500-1:2000 for Western blot and 1:50-1:500 for IHC, which can serve as starting points for biotin-conjugated versions .

What factors affect the reproducibility of results when using biotinylated CLCA1 in experimental systems?

Several critical factors influence the reproducibility of results when using biotinylated CLCA1 in experimental systems. Storage conditions significantly impact antibody and protein stability - biotinylated CLCA1 proteins and antibodies should generally be stored at -20°C and are typically stable for one year after shipment, with aliquoting being unnecessary for -20°C storage . The buffer composition used for storing biotinylated proteins is also crucial; PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is recommended for many antibody preparations . When conducting experiments with biotinylated N-CLCA1, the protein concentration and incubation time are critical variables - typical effective concentrations range from 10-50 μg/ml with 24-hour incubation periods prior to functional assays . Cell culture conditions, particularly for HEK293T cells commonly used in CLCA1 functional studies, must be standardized for consistent results . Additionally, the quality of the biotinylation process itself affects reproducibility - ensure complete removal of excess biotin using appropriate desalting methods to avoid interference with downstream streptavidin binding . Finally, sample-dependent factors should be considered, as noted in the documentation that results may be "sample-dependent," suggesting that different tissue or cell types may require optimization of protocols for consistent results .

How is biotinylated CLCA1 used to investigate calcium-dependent chloride channel regulation?

Biotinylated CLCA1, particularly the N-terminal fragment, serves as a valuable tool for investigating calcium-dependent chloride channel regulation. Researchers can apply purified biotinylated N-CLCA1 (10-50 μg/ml) to cells and assess calcium-dependent chloride currents (ICaCC) via whole-cell patch clamp electrophysiology . These experiments have revealed that secreted CLCA1 activates calcium-dependent chloride currents in a paracrine fashion, with endogenous TMEM16A/Anoctamin1 conducting these currents . When conducting these experiments, intracellular Ca²⁺ concentration (typically 10 μM) and physiological concentrations of extracellular Cl⁻ are maintained to observe robust, slightly outward rectifying currents . Biotinylated CLCA1 can also be used in surface binding experiments to demonstrate that CLCA1 physically engages TMEM16A on the cell surface, leading to increased cell surface levels of TMEM16A . This interaction represents a unique mechanism of channel regulation by a secreted protein. Through these applications, biotinylated CLCA1 has helped establish CLCA1 as the first secreted direct modifier of TMEM16A activity, providing significant insights into the physiology and pathophysiology of airways and other tissues .

What are the implications of CLCA1-TMEM16A interactions for respiratory disease research?

The discovery that CLCA1 directly interacts with and modulates TMEM16A has significant implications for respiratory disease research. Both CLCA1 and TMEM16A have been separately observed to play critical roles in chronic inflammatory airway diseases such as asthma, COPD, and cystic fibrosis . CLCA1 stabilizes TMEM16A on the cell surface, increasing its surface expression and thereby enhancing calcium-dependent chloride currents . This mechanism directly affects mucus production and airway function, which are central to the pathophysiology of these respiratory conditions. By understanding the molecular details of this interaction using biotinylated CLCA1 in research, scientists can explore targeted therapeutic approaches that modulate either CLCA1 secretion or its interaction with TMEM16A . The cooperative partnership between these proteins suggests that they may serve as dual targets for interventions aimed at controlling excessive mucus production and improving airway clearance in patients with chronic inflammatory respiratory diseases . Furthermore, this research has broader implications for other tissues and organs where these proteins are expressed, potentially extending therapeutic applications beyond respiratory conditions .

How might biotinylated CLCA1 antibodies be utilized in high-throughput screening approaches?

Biotinylated CLCA1 antibodies offer unique advantages for high-throughput screening approaches across multiple research applications. In flow cytometry-based screening assays, biotinylated N-CLCA1 pre-incubated with streptavidin-fluorophore conjugates can be used to screen large cell populations for TMEM16A expression levels or to identify compounds that modulate the CLCA1-TMEM16A interaction . For protein-protein interaction studies, biotinylated CLCA1 can be immobilized on streptavidin-coated surfaces (such as microplates or biosensor chips) for screening potential binding partners or small molecule modulators of known interactions. In high-content imaging approaches, biotinylated CLCA1 antibodies can be used with streptavidin-fluorophore conjugates to visualize the cellular localization of CLCA1 across large sets of cells under different treatment conditions. Additionally, biotinylated CLCA1 can be employed in multiplexed assays where multiple biotinylated proteins are detected simultaneously using different streptavidin-conjugated reporters. For drug discovery applications, biotinylated CLCA1-based screening systems could identify compounds that specifically modulate CLCA1's ability to enhance TMEM16A surface expression, potentially leading to novel therapeutics for respiratory diseases characterized by excessive mucus production .

What experimental designs best demonstrate CLCA1's paracrine signaling effects?

To effectively demonstrate CLCA1's paracrine signaling effects, several experimental designs have proven particularly valuable. Co-culture experiments represent a powerful approach: GFP-expressing cells are co-cultured overnight with cells transfected with either CLCA1-pHLsec plasmid or empty pHLsec vector (as control), followed by whole-cell patch clamp electrophysiology to measure calcium-dependent chloride currents in the GFP-positive cells . This design directly demonstrates that CLCA1-expressing cells can activate chloride currents in neighboring cells. Complementary to this, conditioned medium experiments involve culturing untransfected cells in medium obtained from either CLCA1- or vector-transfected cells, followed by patch clamp measurements . This approach confirms that secreted CLCA1 in the medium is sufficient for current activation. For mechanistic studies, cellular binding assays using biotinylated N-CLCA1 coupled to streptavidin-phycoerythrin create fluorescent tetrameric reagents with enhanced avidity . Flow cytometry analysis of binding in the presence or absence of blocking antibodies against potential interaction partners (such as TMEM16A) helps elucidate the molecular mechanisms of CLCA1's paracrine effects . Combined, these experimental designs provide complementary evidence for CLCA1's role as a secreted modifier of calcium-dependent chloride channel activity.

How should researchers interpret conflicting results between different detection methods when studying CLCA1?

When researchers encounter conflicting results between different detection methods for CLCA1, systematic troubleshooting and careful interpretation are required. First, consider the specific epitopes targeted by different antibodies or detection reagents. CLCA1 undergoes autocatalytic cleavage, resulting in multiple fragments—the observed molecular weight (50 kDa) differs significantly from the calculated weight (100 kDa) . Different antibodies may preferentially detect particular fragments, leading to apparently conflicting results. Second, evaluate the impact of sample preparation methods on CLCA1 detection. Certain fixatives or buffers may alter epitope accessibility or protein conformation, affecting antibody binding differently across methods . For IHC applications, antigen retrieval methods significantly impact results—TE buffer pH 9.0 is recommended for CLCA1 detection, though citrate buffer pH 6.0 is an alternative . Third, assess assay sensitivity differences—flow cytometry, Western blotting, and immunohistochemistry have inherently different detection limits and dynamic ranges. Finally, consider biological variables—CLCA1 expression and processing may vary between tissue types and experimental conditions. When reporting conflicting results, researchers should comprehensively document methodological details including antibody clones, detection methods, sample preparation protocols, and statistical analyses to facilitate interpretation and reproducibility of findings.