Recombinant Human CLC

Using Recombinant Human CLC (Chloride Channel protein) in research applications enables precise investigation of chloride channel function, disease mechanisms, and drug screening, thanks to its high purity, reproducibility, and customizable properties.

Key scientific advantages:

• Functional Studies: Recombinant human CLC proteins allow direct characterization of chloride channel activity, ion selectivity, gating mechanisms, and regulatory features in controlled systems. This is essential for understanding physiological roles such as cell volume regulation, transepithelial transport, and electrical excitability.

• Disease Modeling: Mutations in CLC genes are linked to various human diseases, including neuromuscular and renal disorders. Recombinant CLC enables modeling of disease-relevant mutations and assessment of their impact on channel function, facilitating mechanistic studies and therapeutic target validation.

• Drug Discovery: Recombinant CLC proteins are used in high-throughput screening assays to identify and characterize compounds that modulate chloride channel activity, supporting the development of targeted therapies for channelopathies and other conditions.

• Structural Biology: Large-scale production of recombinant CLC is critical for structural studies (e.g., X-ray crystallography, cryo-EM), which reveal channel architecture, conformational changes, and drug binding sites.

• Consistency and Safety: Recombinant proteins offer superior batch-to-batch consistency, defined molecular composition, and eliminate risks associated with animal-derived materials (e.g., pathogen transmission, immunogenicity).

• Customizability: Recombinant expression systems allow for engineering of specific mutations, tags, or fusion partners, enabling tailored experimental designs for mechanistic, localization, or interaction studies.

Typical research applications include:

• Electrophysiological assays (patch-clamp, voltage-clamp) to measure channel currents and kinetics.
• Structure-function analysis of wild-type and mutant CLC proteins.
• Screening and validation of pharmacological modulators.
• Development of diagnostic assays for channelopathies.
• Comparative studies with native chloride channels to elucidate physiological and pathological mechanisms.

In summary, Recombinant Human CLC is an indispensable tool for rigorous, reproducible, and mechanistic research on chloride channels, with broad utility in physiology, disease modeling, drug discovery, and structural biology.

Yes, recombinant human CLC can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly reconstituted. This is a common practice in quantitative ELISA, where a purified or recombinant protein is used to generate a standard curve against which unknown sample concentrations are measured.

Key considerations and best practices:

• Purity and Formulation: The recombinant CLC should be of high purity (typically >90% as determined by SDS-PAGE) and free from contaminants that could interfere with antibody binding or detection. Many recombinant CLC proteins are supplied lyophilized and may include a carrier protein such as BSA to enhance stability, which is generally suitable for ELISA standards.

• Reconstitution: Follow the manufacturer’s instructions for reconstitution, as the buffer composition and presence of carrier proteins can affect protein stability and assay performance. For example, some protocols recommend reconstituting in 4 mM HCl with at least 0.1% serum albumin.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant CLC to cover the expected concentration range in your samples (commonly 0–1000 pg/mL, but this may vary depending on your assay sensitivity and sample type). Each ELISA plate should include its own standard curve for accurate quantification.

• Validation: It is important to validate that the recombinant CLC standard behaves similarly to the endogenous protein in your assay. Differences in glycosylation, folding, or source (e.g., E. coli vs. mammalian expression) can sometimes affect antibody recognition or assay linearity. If possible, spike recombinant CLC into your sample matrix to assess recovery and parallelism.

• Assignment of Standard Value: When using a recombinant protein as a standard, it is best to assign its value based on its performance in the ELISA rather than relying solely on the mass stated on the vial, as immunoreactivity may differ between lots or preparations.

Summary Table: Key Requirements for Using Recombinant CLC as an ELISA Standard

In summary: Recombinant human CLC is suitable as an ELISA standard if it is pure, properly reconstituted, and validated in your assay system. Always include a standard curve on each plate and confirm that the recombinant standard provides accurate quantification in your specific assay context.

Recombinant Human CLC has been validated for several key applications in published research, primarily in bioassays, cell culture, and studies of cell signaling and protein-protein interactions.

• Bioassay: Recombinant Human CLC protein is routinely used in bioactivity assays to study its functional properties, including its ability to bind with cytokine-like factor-1 (CLF) and initiate downstream signaling via gp130-LIF R dimerization. These assays help characterize the biological activity and potency of the protein.

• Cell Culture: The protein is applied in cell culture systems to investigate its effects on cellular responses, such as differentiation, proliferation, or signaling. For example, it has been used to study gene regulatory networks in human cells, including applications in heart regeneration research.

• Protein-Protein Interaction Studies: CLC is known to form heterodimeric complexes with CLF, which are critical for cytokine signaling pathways. Recombinant CLC is used to dissect these interactions and their functional consequences in vitro.

• Mechanistic Studies of Signaling: Research has validated recombinant CLC for exploring its role in activating membrane-associated receptors (e.g., CNTF R alpha) and subsequent intracellular signaling cascades.

• Sample Types: Validated sample types include whole cells, indicating utility in both cell-based and biochemical assays.

Additional Context:

• The protein is typically expressed in E. coli and may be supplied with or without carrier proteins (e.g., BSA), depending on the intended application (cell culture vs. ELISA standard).
• While some sources mention use in ELISA, direct validation for ELISA as a standard is less frequently cited in published research compared to bioassays and cell culture applications.

Summary Table of Validated Applications

If you require details on specific experimental protocols or additional applications (e.g., ELISA, imaging, or therapeutic studies), please specify, as published research primarily validates recombinant human CLC for the above core uses.

To properly reconstitute and prepare Recombinant Human CLC protein for cell culture experiments, follow these best practices based on manufacturer guidelines and general recombinant protein handling protocols:

1. Reconstitution

• Centrifuge the vial briefly before opening to ensure the lyophilized powder is at the bottom.
• Reconstitute at 100 µg/mL in sterile 4 mM HCl (as recommended for E. coli-derived CLC protein).
• If the product datasheet or certificate of analysis specifies the addition of a carrier protein, include at least 0.1% human or bovine serum albumin (BSA) in the reconstitution buffer to stabilize the protein.
• Gently mix the solution by swirling or slow inversion; avoid vigorous shaking to prevent foaming and protein denaturation.
• Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

2. Dilution for Cell Culture

• After reconstitution, dilute the stock solution in your cell culture medium or an appropriate buffer.
• For most bioassays and cell culture applications, typical working concentrations range from 3–15 ng/mL.
• If using serum-free or low-protein media, consider adding 0.1% BSA or 1–10% FBS to the final culture medium to help stabilize the protein and prevent adsorption to plastic surfaces.

3. Storage

• Short-term storage: Store the reconstituted protein at 2–8°C for up to one month if used frequently.
• Long-term storage: Aliquot the protein solution and store at –20°C to –80°C. Avoid repeated freeze-thaw cycles to maintain activity.
• For extended storage, consider adding 5–50% glycerol (final concentration) to the aliquots before freezing.

4. General Tips

• Always refer to the product datasheet or certificate of analysis for lot-specific instructions.
• Use sterile techniques throughout to prevent contamination.
• For sensitive cell types, pre-test the protein for cytotoxicity or non-specific effects at the intended working concentration.

By following these steps, you will ensure optimal activity and stability of Recombinant Human CLC protein in your cell culture experiments.