EFCAB14 Antibody

What is EFCAB14 and what cellular functions does it perform?

EFCAB14, also known as KIAA0494, is a 495 amino acid protein that contains two EF-hand domains—helix-loop-helix structures typically found in calcium-binding proteins . The gene encoding EFCAB14 maps to human chromosome 1p33 . While the complete functional characterization of EFCAB14 remains ongoing, its EF-hand domains suggest involvement in calcium-dependent cellular processes.

The protein's molecular weight is approximately 55 kDa, which is important to note when validating antibody specificity in western blot applications . As a calcium-binding protein, EFCAB14 potentially participates in calcium signaling pathways, which regulate diverse cellular processes including gene transcription, cell proliferation, and apoptosis. Current research continues to elucidate the specific pathways and protein interactions involving EFCAB14.

What types of EFCAB14 antibodies are available for research applications?

Several types of EFCAB14 antibodies are available for research, primarily polyclonal antibodies raised in rabbits:

• Rabbit polyclonal antibodies that target human EFCAB14 (0.2 mg/ml concentration)

• Rabbit polyclonal antibodies validated for human and rat samples (0.59 mg/ml concentration)

• Rabbit polyclonal antibodies raised against recombinant human EF-hand calcium-binding domain-containing protein 14 (1-483AA)

These antibodies have been validated for various applications including Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) . The availability of different formats allows researchers to select antibodies optimized for their specific experimental approaches.

How should EFCAB14 antibodies be stored to maintain optimal activity?

Proper storage of EFCAB14 antibodies is critical for maintaining reactivity and specificity. The recommended storage conditions are:

• Aliquot the antibody upon receipt to minimize freeze-thaw cycles

• Store at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles that can degrade the antibody and reduce performance

Some antibodies are supplied in stabilizing buffers (e.g., "0.1M Tris (pH 7.0), 0.1M Glycine and 10% Glycerol with 0.01% Thimerosal") that help maintain activity. When planning long-term research projects, it's advisable to create multiple small aliquots rather than repeatedly accessing a single stock, as each freeze-thaw cycle can diminish antibody performance by approximately 10-15%.

What are the optimal dilution ratios for EFCAB14 antibodies in different applications?

The optimal dilution ratios for EFCAB14 antibodies vary by application technique and specific antibody manufacturer. Based on the available data, the following dilution ranges are recommended:

These ranges serve as starting points, and optimization is recommended for each specific experimental system. Factors affecting optimal dilution include antibody batch, sample type, target abundance, and detection system sensitivity. A dilution series experiment is advised when using the antibody for the first time in a particular application.

What sample preparation methods are recommended for detecting EFCAB14 in different sample types?

Effective sample preparation is crucial for successful detection of EFCAB14 in various sample types:

For cellular/tissue lysates:

• Use RIPA buffer (containing protease inhibitors) for most applications

• Lyse cells on ice for 30 minutes with periodic vortexing

• Centrifuge at 14,000 × g for 15 minutes at 4°C to remove cell debris

• Protein concentration should be determined using Bradford or BCA assay

For serum/plasma samples:

• Maximum sample volume recommended is 50 μl per reaction

• Dilute samples 1:2 to 1:5 with sample diluent to minimize matrix effects

• For ELISA applications, avoid using hemolyzed or lipemic samples

For cell culture supernatants:

• Collect supernatants and centrifuge to remove cell debris

• Up to 100 μl can be used per reaction in ELISA applications

• Serum-free media is preferred to avoid interference from serum proteins

When preparing samples for Western blot, include a reducing agent in the sample buffer and heat at 95°C for 5 minutes to ensure proper denaturation for optimal epitope exposure.

What controls should be included when using EFCAB14 antibodies for experimental validation?

Proper experimental controls are essential for validating results obtained with EFCAB14 antibodies:

• Positive control: Samples known to express EFCAB14 (specific cell lines or tissues)

• Negative control: Samples known not to express EFCAB14 or with EFCAB14 expression knocked down

• Primary antibody control: Omitting the primary antibody while maintaining all other steps

• Isotype control: Using a non-specific antibody of the same isotype and host species

• Blocking peptide control: Pre-incubating the antibody with an excess of the immunogen peptide

For knockdown validation, comparing EFCAB14 antibody reactivity between wildtype and EFCAB14 siRNA/shRNA-treated samples provides strong evidence of specificity. Additionally, using multiple antibodies targeting different epitopes of EFCAB14 can further confirm specificity and reduce the risk of misinterpreting results due to cross-reactivity.

How can EFCAB14 antibodies be utilized in co-immunoprecipitation experiments to study protein-protein interactions?

Co-immunoprecipitation (Co-IP) using EFCAB14 antibodies can reveal physiologically relevant protein interactions:

• Sample preparation:

  • Prepare cell/tissue lysates using a non-denaturing lysis buffer (e.g., 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA) with protease inhibitors

  • Maintain samples at 4°C throughout to preserve protein-protein interactions

• Pre-clearing step:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation to reduce non-specific binding

• Immunoprecipitation:

  • Add 2-5 μg of EFCAB14 antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add Protein A/G beads and incubate for 2-4 hours

  • Wash beads 4-5 times with lysis buffer

• Analysis:

  • Elute bound proteins by boiling in SDS sample buffer

  • Analyze by SDS-PAGE followed by western blotting for potential interacting partners

When investigating calcium-dependent interactions, consider performing parallel experiments with varying calcium concentrations (using EGTA for calcium chelation or CaCl₂ for calcium supplementation) to identify calcium-dependent binding partners of EFCAB14.

What approaches can be used to study EFCAB14 localization and trafficking in live cells?

Studying EFCAB14 localization and trafficking in live cells requires specialized approaches:

• Antibody-based live-cell imaging:

  • Use cell-permeable fluorescently-tagged Fab fragments derived from EFCAB14 antibodies

  • Alternatively, microinject fluorescently labeled EFCAB14 antibodies

• Fusion protein approaches:

  • Generate EFCAB14-GFP (or other fluorescent protein) fusion constructs

  • Transfect cells and perform time-lapse confocal microscopy

  • Consider photoactivatable or photoswitchable fluorescent proteins for pulse-chase experiments

• Calcium dynamics integration:

  • Combine EFCAB14-fluorescent protein imaging with calcium indicators (e.g., Fluo-4)

  • Analyze EFCAB14 trafficking in response to calcium flux using stimuli like ionomycin

• Super-resolution approaches:

  • Apply techniques like STORM or PALM using antibodies against EFCAB14

  • These methods can resolve structures below the diffraction limit, potentially revealing novel subcellular localizations

When designing these experiments, consider using the calcium ionophore ionomycin or other stimuli that alter intracellular calcium levels to determine whether EFCAB14 localization changes in response to calcium fluctuations, which would be expected for a calcium-binding protein.

How can EFCAB14 antibodies be employed in chromatin immunoprecipitation (ChIP) studies?

Although EFCAB14 is not primarily characterized as a DNA-binding protein, investigating its potential association with chromatin or chromatin-associated proteins can be approached through:

• Standard ChIP protocol:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Lyse cells and sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate using 3-5 μg of EFCAB14 antibody

  • Reverse crosslinks and analyze DNA by qPCR or sequencing

• Sequential ChIP (Re-ChIP):

  • Perform initial ChIP with antibodies against known chromatin-associated proteins

  • Elute complexes under non-denaturing conditions

  • Perform second ChIP with EFCAB14 antibodies

  • This approach identifies regions where EFCAB14 co-localizes with specific chromatin factors

• ChIP-MS (ChIP followed by mass spectrometry):

  • Use EFCAB14 antibodies for ChIP

  • Instead of analyzing DNA, identify co-precipitated proteins by mass spectrometry

  • This approach characterizes the protein complexes associated with EFCAB14 at chromatin

These approaches would be particularly relevant if preliminary data suggests nuclear localization of EFCAB14 or interactions with transcription factors, which would be consistent with the calcium-dependent regulatory functions of many EF-hand domain-containing proteins.

What are common causes of non-specific binding with EFCAB14 antibodies and how can they be addressed?

Non-specific binding is a common challenge when working with antibodies. For EFCAB14 antibodies:

• Sources of non-specific binding:

  • Cross-reactivity with related EF-hand domain-containing proteins

  • Insufficient blocking

  • Excessive antibody concentration

  • Sample overloading

• Optimization strategies:

  • Increase blocking time/concentration (5% BSA or milk can be more effective than 3%)

  • Reduce primary antibody concentration (try a dilution series)

  • Add 0.1-0.5% Tween-20 to washing buffers to reduce hydrophobic interactions

  • Pre-adsorb antibody with acetone powder from tissues lacking EFCAB14

• Validation approaches:

  • Compare results using multiple EFCAB14 antibodies targeting different epitopes

  • Include EFCAB14 knockdown/knockout controls

  • Use blocking peptide competition to identify specific bands

For western blotting specifically, remember that EFCAB14 has a molecular weight of approximately 55 kDa . Bands at significantly different molecular weights likely represent non-specific binding or post-translationally modified forms of the protein, which should be validated through additional experiments.

How can researchers validate the specificity of their EFCAB14 antibody detection in experimental systems?

Validating antibody specificity is crucial for reliable research outcomes:

• Genetic approaches:

  • Compare antibody reactivity in wildtype versus EFCAB14 knockdown/knockout systems

  • Use siRNA, shRNA, or CRISPR-Cas9 to reduce EFCAB14 expression

  • Expected result: Reduced or eliminated signal in knockdown/knockout samples

• Recombinant protein controls:

  • Test antibody reactivity against purified recombinant EFCAB14

  • Include related EF-hand proteins to assess cross-reactivity

  • Perform peptide competition assays using the immunizing peptide

• Multiple detection methods:

  • Confirm findings using different techniques (e.g., IF, WB, IHC)

  • Use multiple antibodies targeting different EFCAB14 epitopes

  • Compare results with mRNA expression data (RT-qPCR)

• Tissue/cell type expression profiling:

  • Test antibody across a panel of tissues/cell types with known EFCAB14 expression levels

  • Compare protein detection patterns with published transcriptomic data

Comprehensive validation through multiple independent approaches provides the strongest evidence for antibody specificity and should be performed before undertaking extensive experimental work with a new EFCAB14 antibody.

What factors affect the sensitivity of EFCAB14 detection in ELISA assays and how can they be optimized?

ELISA sensitivity for EFCAB14 detection can be affected by multiple factors:

• Key sensitivity determinants:

  • Antibody affinity and specificity

  • Sample preparation and matrix effects

  • Blocking efficiency

  • Enzyme-substrate kinetics

  • Incubation times and temperatures

• Optimization strategies:

  • Sample dilution optimization: Prepare a dilution series to identify optimal concentration range (samples should fall within the linear portion of the standard curve)

  • Antibody concentration: Test different concentrations of capture and detection antibodies

  • Signal amplification: Consider using streptavidin-HRP systems or tyramide signal amplification

  • Extended substrate incubation: Allow color development to proceed longer for low-abundance samples

• Performance metrics:

  • The typical detection range for EFCAB14 ELISA kits is 78.125-5000 pg/ml

  • Sensitivity (lower limit of detection) is approximately 46.875 pg/ml

  • Review standard curve parameters (r² value should exceed 0.98)

When working with complex samples like serum or cell lysates, consider sample pre-treatment methods such as heat inactivation, filtration, or pre-adsorption with irrelevant proteins to reduce matrix effects that might interfere with EFCAB14 detection.