calhm5.1 Antibody

What is CALHM5 and what cellular functions does it perform?

CALHM5 (calcium homeostasis modulator family member 5) is a membrane-localized protein that functions as a pore-forming subunit of voltage-gated ion channels. In humans, the canonical protein has 309 amino acid residues and a molecular weight of approximately 35.2 kDa . It belongs to the CALHM protein family, which includes several members with roles in calcium homeostasis and ion transport.

While specific CALHM5 functions remain under investigation, research on related family members provides valuable insights. For instance, CALHM1 has been extensively studied in neuronal contexts and shown to regulate amyloid-β (Aβ) clearance via control of insulin-degrading enzyme (IDE) secretion . CALHM proteins more broadly have been investigated in placental tissues, suggesting diverse physiological roles .

CALHM5 is also known by alternate names including:

• Family with sequence similarity 26 member E (FAM26E)

• Calcium homeostasis modulator protein 5

What applications are CALHM5 antibodies validated for in research settings?

CALHM5 antibodies are validated for multiple applications in molecular and cellular research:

Researchers should select antibodies specifically validated for their intended application, as performance may vary significantly between applications .

What species reactivity is available for CALHM5 antibodies?

Commercial CALHM5 antibodies demonstrate variable cross-reactivity across species:

CALHM5 gene orthologs have been reported in mouse, rat, bovine, frog, chimpanzee, and chicken species , providing opportunities for comparative studies across these organisms.

How should researchers validate the specificity of CALHM5 antibodies?

Rigorous validation of CALHM5 antibodies is essential for experimental reliability. Recommended validation approaches include:

• Western blot analysis: Confirm detection of a band at the expected molecular weight (35.2 kDa for human CALHM5) . Include positive and negative controls to verify specificity.

• Genetic validation: Compare antibody signals between wild-type samples and those with CALHM5 knockdown/knockout to confirm specificity.

• Epitope mapping: Understand which region of CALHM5 the antibody recognizes. Several commercial antibodies target the middle region of CALHM5 , which may affect detection of specific splice variants or modified forms.

• Cross-reactivity assessment: Test for potential cross-reactivity with other CALHM family members, particularly important when studying tissues expressing multiple CALHM proteins.

• Protein array validation: Some antibodies undergo specificity analysis using protein arrays to verify target binding . This provides additional confidence in antibody specificity.

When selecting antibodies, researchers should prioritize those with documented validation data for their specific application and experimental system.

What are effective protocols for studying CALHM5 expression at the mRNA level?

For quantitative analysis of CALHM5 transcript expression:

• Primer design: Design specific primers that distinguish CALHM5 from other CALHM family members. Consider:

  • Targeting unique exon junctions

  • Testing primer efficiency using standard curves

  • Confirming specificity through melt curve analysis

• Reference gene selection: YWHAZ (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) has been successfully used as a reference gene in CALHM expression studies .

• qPCR protocol: A validated protocol involves:

  • Reaction mix: 0.5 μM primers, 2× qPCR Master Mix, and 1 μl cDNA in 10 μl total volume

  • Amplification in duplicate reactions

  • Data analysis using the 2^-ΔΔCt method for relative quantification

• Controls: Include no-template controls, no-reverse transcriptase controls, and positive controls from tissues known to express CALHM5.

What experimental considerations are important when performing Western blot analysis for CALHM5?

As a membrane protein, CALHM5 requires specific considerations for effective Western blot analysis:

• Sample preparation:

  • Effective lysis buffers include those containing LMNG (1%) or GDN (2%)

  • Include divalent cations (0.5 mM CaCl₂, 2 mM MgCl₂) for protein stability

  • For calcium-free studies, substitute with 5 mM EGTA

• Gel electrophoresis:

  • Use gradient gels (4-15%) for optimal separation of membrane proteins

  • Heat samples at 37°C rather than boiling to prevent aggregation

• Transfer conditions:

  • Optimize transfer time and voltage for efficient membrane transfer

  • Consider semi-dry transfer systems for membrane proteins

• Detection:

  • Primary antibody dilutions typically range from 1:1000 to 1:5000

  • Extended incubation times (overnight at 4°C) may improve signal quality

  • Use fluorescent secondary antibodies for quantitative analysis

• Controls:

  • Include positive controls (tissues/cells known to express CALHM5)

  • Use recombinant CALHM5 as a molecular weight reference when available

How can functional studies of CALHM5 ion channel activity be designed?

For investigating the ion channel properties of CALHM5:

• Expression systems: Several systems have proven effective for CALHM family proteins:

  • HEK293T cells for screening experiments

  • HEK293S GnTI- cells for protein purification

  • Xenopus laevis oocytes for electrophysiological studies

• Transfection approaches:

  • Polyethylenimine (PEI) transfection has been effective for CALHM expression

  • For HEK293T cells: 12 μg DNA and 48 μg PEI in 0.5 ml DMEM medium

  • For suspension cultures: 0.5 mg DNA and 1.2 mg PEI MAX in 20 ml DMEM, supplemented with 4 mM valproic acid

• Electrophysiological methods:

  • Patch-clamp recordings to measure conductance, kinetics, and voltage sensitivity

  • Ion substitution experiments to determine selectivity profiles

  • Pharmacological characterization using channel blockers/modulators

• Calcium imaging:

  • Using calcium-sensitive dyes or genetically encoded calcium indicators

  • Testing in calcium-free conditions (with EGTA) as a control

  • Measuring response kinetics under various stimulation conditions

What approaches are recommended for structural studies of CALHM5?

For structural investigations of CALHM5:

• Protein purification protocol:

  • Cell lysis in buffer containing 25 mM HEPES, 150 mM NaCl, 1% LMNG, 0.5 mM CaCl₂, 2 mM MgCl₂, protease inhibitors, RNase, DNase, pH 7.6

  • Incubation for 1 hour under constant stirring at 4°C

  • For calcium-free conditions, substitute calcium with 5 mM EGTA

• Chromatography methods:

  • Size exclusion chromatography using Superose 6 columns for analysis of oligomeric states

  • Equilibration in buffer containing 10 mM HEPES, 150 mM NaCl, 50 μM GDN, pH 7.6

• Structural analysis techniques:

  • Cryo-electron microscopy (cryo-EM) has been successfully applied to CALHM proteins

  • Negative stain EM for initial structural assessment

  • Biochemical crosslinking combined with mass spectrometry for interaction mapping

• Fluorescent tagging approaches:

  • C-terminal Venus-Myc-SBP tags have been successfully used with CALHM proteins

  • Tagging with 3C protease cleavage sites allows tag removal after purification

What can be inferred about CALHM5 function based on studies of related CALHM proteins?

While specific CALHM5 functions require further investigation, research on related family members suggests potential roles:

• Calcium signaling: CALHM1 forms a plasma membrane Ca²⁺ channel , suggesting CALHM5 may similarly participate in calcium homeostasis pathways.

• Amyloid-β regulation: CALHM1 controls amyloid-β levels through:

  • Promoting extracellular Aβ degradation

  • Enhancing insulin-degrading enzyme (IDE) secretion

  • Requiring ion permeability and extracellular calcium for this effect

• Pathological implications: CALHM1 variants have been associated with Alzheimer's disease risk and altered amyloid-β levels in human cerebrospinal fluid . This suggests potential roles for CALHM family members, possibly including CALHM5, in neurological conditions.

• Placental physiology: CALHM proteins have been investigated in placental tissues, with expression patterns changing during trophoblast differentiation , suggesting potential roles in pregnancy and development.

What are common challenges when working with CALHM5 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when using CALHM5 antibodies:

• Low signal strength:

  • Problem: Weak detection of endogenous CALHM5

  • Solution: Optimize antibody concentration; use signal enhancement systems; consider tissues with higher expression levels

• Membrane protein extraction:

  • Problem: Incomplete solubilization of membrane-embedded CALHM5

  • Solution: Use effective detergents like LMNG (1%) or GDN (2%); avoid harsh denaturing conditions for conformational epitopes

• Cross-reactivity concerns:

  • Problem: Non-specific binding to other CALHM family members

  • Solution: Select antibodies targeting unique epitopes; validate using knockout controls; perform comprehensive blocking experiments

• Background in immunostaining:

  • Problem: High non-specific staining in ICC/IHC applications

  • Solution: Optimize blocking conditions (5% BSA or 10% serum); titrate primary antibody; include appropriate isotype controls

• Batch-to-batch variability:

  • Problem: Performance differences between antibody lots

  • Solution: Maintain reference samples; document lot numbers; consider monoclonal antibodies for greater consistency

How can gene expression analysis of CALHM5 be optimized?

For robust analysis of CALHM5 expression:

• RNA isolation optimization:

  • Use specialized kits for tissues with high RNase activity

  • Process samples rapidly and maintain cold conditions

  • Include RNase inhibitors during extraction

• RT-qPCR considerations:

  • Design primers with efficiency between 90-110%

  • Target primer concentration: 0.5 μM

  • Use validated reference genes like YWHAZ

  • Calculate relative expression using 2^-ΔΔCt method

• Expression comparison protocol:

  • A validated approach for comparing expression between cell states (e.g., undifferentiated CTB vs differentiated STB cells):

    • Calculate ΔCt = (Ct value of reference gene - Ct value of CALHM5)

    • Determine ΔΔCt between conditions

    • Express as fold change using 2^-ΔΔCt

    • Analyze using paired 2-way ANOVA with Sidak's multiple comparisons test

• Controls and validation:

  • Include positive control tissues with known CALHM5 expression

  • Verify primer specificity through sequencing of PCR products

  • Use both biological and technical replicates (minimum triplicate biological samples)

What key experimental controls should be included in CALHM5 functional studies?

For rigorous experimental design in CALHM5 functional studies:

• Expression verification controls:

  • Confirm CALHM5 expression in experimental system via Western blot or qPCR

  • Include vector-only transfection controls in overexpression studies

  • For tagged proteins, verify that tags don't interfere with function

• Calcium modulation controls:

  • Include calcium-free conditions (with EGTA) to test calcium dependency

  • Use known calcium channel modulators as positive controls

  • Test multiple calcium concentrations (e.g., 0.2 mM vs. standard levels)

• Genetic controls:

  • Compare wild-type to CALHM5 knockout/knockdown models

  • Include rescue experiments with wild-type CALHM5 to confirm specificity

  • Use domain mutants to investigate structure-function relationships

• Physiological relevance controls:

  • Compare results across multiple cell types with different endogenous CALHM5 levels

  • Validate key findings in primary cells when possible

  • Consider developmental timing and physiological state in interpretation