FAM234A Antibody, Biotin conjugated

What is the FAM234A protein and why is it studied?

FAM234A (also known as ITFG3 or C16orf9) is a human protein with the UniProt ID Q9H0X4 . This protein is primarily studied in cell biology research contexts, with increasing interest in its functional roles in cellular processes . FAM234A has a molecular weight of approximately 60kDa and contains specific peptide sequences of interest including "RSAFFFWGLHELGSTSETETGEARHSLYMFHPTLPRVLLELANVSTHIVA" that are often targeted for antibody development . The research interest in FAM234A stems from its potential involvement in cellular signaling pathways, though comprehensive functional characterization remains an ongoing research area. Researchers typically use antibodies against FAM234A to investigate its expression patterns, subcellular localization, and potential protein-protein interactions in human cell systems .

What are the key applications for FAM234A Antibody, Biotin conjugated?

The primary application documented for commercially available FAM234A Antibody, Biotin conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . This specificity makes it particularly valuable for quantitative detection of FAM234A in human samples. The biotin conjugation enables signal amplification when used with streptavidin or avidin detection systems, which is especially useful for detecting proteins expressed at low levels . While ELISA is the validated application, the antibody may potentially be suitable for other techniques that utilize biotinylated antibodies, such as immunohistochemistry (IHC) or western blotting, though researchers should conduct validation studies before applying to these methods . The specificity for human samples (rather than cross-reactivity with other species) makes this antibody ideal for human cell line research and clinical sample analysis .

How does biotin conjugation benefit detection of FAM234A compared to other conjugates?

Biotin conjugation offers several significant advantages for FAM234A detection compared to other conjugation methods. First, the biotin-streptavidin binding system provides one of the strongest non-covalent interactions known in biology, with remarkably high affinity (Kd ≈ 10^-15 M), ensuring stable and specific detection . Second, each antibody molecule can be conjugated with multiple biotin molecules, creating opportunity for significant signal amplification when combined with avidin/streptavidin detection systems . This amplification is particularly valuable for FAM234A research where protein expression might be low or in samples with limited availability. Additionally, biotin-conjugated antibodies offer flexibility in detection methods, as they can be visualized using streptavidin conjugated to various reporter molecules (fluorescent dyes, enzymes, etc.), allowing researchers to adapt their detection strategy based on available equipment and experimental needs . Compared to direct fluorophore conjugation (like FITC), biotin conjugation generally provides superior signal-to-noise ratio and detection sensitivity for low-abundance proteins like FAM234A .

What are the optimal storage and handling conditions for FAM234A Antibody, Biotin conjugated?

The optimal storage and handling conditions for FAM234A Antibody, Biotin conjugated are critical for maintaining its activity and specificity. The antibody should be stored at -20°C or -80°C for long-term preservation, and it's important to avoid repeated freeze-thaw cycles that can degrade antibody quality . For short-term storage (up to 2 weeks), refrigeration at 2-8°C is acceptable . The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during storage. When handling the antibody, it's advisable to aliquot the stock solution into smaller volumes upon receipt to minimize freeze-thaw cycles . Working dilutions should be prepared fresh before use and kept at 4°C for short periods only. Laboratory protocols should include careful temperature monitoring during handling, as biotin conjugates can be sensitive to extended periods at room temperature. Documentation of storage conditions, freeze-thaw cycles, and handling procedures is recommended as part of good laboratory practice for troubleshooting potential experimental issues .

What is the recommended protocol for using FAM234A Antibody, Biotin conjugated in ELISA?

When using FAM234A Antibody, Biotin conjugated in ELISA applications, researchers should follow a methodical approach for optimal results. Begin by coating the ELISA plate with your capture antibody or antigen, depending on whether you're performing a sandwich ELISA or direct ELISA. After blocking with an appropriate blocking buffer (typically containing BSA or serum), add your samples containing the FAM234A protein. The biotinylated FAM234A antibody should be diluted according to the manufacturer's recommendations, which may suggest optimization between applications . After incubation with the biotinylated antibody and washing steps, introduce streptavidin conjugated to an enzyme such as horseradish peroxidase (HRP). The streptavidin will bind with high affinity to the biotin on the antibody, providing a detection system when an appropriate substrate is added. For quantitative analysis, include a standard curve using recombinant FAM234A protein at known concentrations. The optimal antibody concentration should be determined empirically, but manufacturers typically suggest a starting concentration for ELISA applications. For instance, some protocols recommend antibody titration starting at 0.2-1 μg/ml with an expected ELISA titer of approximately 1:62500 . Validation using positive controls such as human MCF7 cell lysate is advised to confirm specificity .

How can researchers verify the specificity of FAM234A Antibody, Biotin conjugated?

Verifying the specificity of FAM234A Antibody, Biotin conjugated requires a multi-faceted approach. First, researchers should examine the antibody's documentation for information about the immunogen used in its production. For FAM234A antibodies, the immunogen is typically recombinant Human Protein FAM234A protein (amino acids 115-251) or a synthetic peptide directed towards the middle region of human ITFG3 . To experimentally validate specificity, researchers should perform western blot analysis using positive control samples known to express FAM234A (such as MCF7 cell lysate) and negative control samples where FAM234A expression is absent or knocked down . The observed band should match the expected molecular weight of approximately 60kDa . Additionally, researchers can perform competitive inhibition assays by pre-incubating the antibody with the immunizing peptide or recombinant protein before application to samples. Specific binding should be blocked in this case. Cross-reactivity testing against samples from other species is less relevant for this particular antibody as it is specifically reactive with human FAM234A . For advanced validation, techniques such as immunoprecipitation followed by mass spectrometry can provide definitive confirmation of target specificity. Researchers should also consider testing for potential cross-reactivity with other members of the FAM protein family to ensure signal specificity in complex biological samples .

How can background signal be minimized when using FAM234A Antibody, Biotin conjugated?

Minimizing background signal when using FAM234A Antibody, Biotin conjugated requires addressing several potential sources of non-specific binding. First, evaluate your blocking protocol, as insufficient blocking is a common cause of background. For FAM234A detection, a blocking buffer containing 1-5% BSA or 5-10% normal serum (from the species in which the secondary detection reagent was raised) in PBS or TBS is typically effective . Second, address endogenous biotin interference, which is particularly important when working with biotin-conjugated antibodies. Using an Endogenous Biotin-Blocking Kit before applying the biotinylated antibody can significantly reduce this source of background . Third, optimize antibody concentration through titration experiments; excess antibody often leads to increased background. For FAM234A antibody, start with the manufacturer's recommended dilution (typically in the range of 0.2-1 μg/ml for ELISA applications) and adjust as needed . Fourth, increase the stringency and number of washing steps using PBS or TBS with 0.05-0.1% Tween-20. Fifth, when using streptavidin detection systems, pre-absorb the streptavidin conjugate with the sample matrix to remove potential cross-reactive components. Finally, consider signal-to-noise enhancement techniques such as incorporating divalent cations (Ca²⁺, Mg²⁺) in wash buffers at 1-2 mM concentration to stabilize specific antibody-antigen interactions while reducing non-specific binding . Systematic optimization of these parameters should be documented to establish reproducible protocols with minimal background.

What strategies can improve signal detection when FAM234A expression is low?

Enhancing signal detection for low-abundance FAM234A requires leveraging the amplification potential of biotin-conjugated antibodies. The primary strategy involves implementing a tyramide signal amplification (TSA) system, which can increase sensitivity by 10-100 fold over conventional detection methods . For biotinylated FAM234A antibody specifically, products like the Biotin XX Tyramide SuperBoost Kit with HRP-conjugated streptavidin can dramatically amplify the signal . This technique deposits multiple biotin molecules at the antibody binding site, which can then be detected with fluorophore-conjugated streptavidin. Second, optimize sample preparation to concentrate FAM234A protein through immunoprecipitation before analysis. Third, extend primary antibody incubation time to 12-18 hours at 4°C to increase binding efficiency without compromising specificity. Fourth, use signal enhancement buffers containing polyvinyl alcohol or dextran sulfate which can increase signal intensity by slowing down the enzymatic reaction in chromogenic detection systems. Fifth, consider using more sensitive detection substrates - for HRP-based detection, enhanced chemiluminescence (ECL) substrates with extended signal duration allow for multiple exposures to optimize signal capture. Finally, address potential protein degradation by incorporating protease inhibitor cocktails in all sample preparation steps and handling samples at 4°C whenever possible . These combined approaches can significantly improve the detection of low-abundance FAM234A while maintaining specificity.

How can researchers address cross-reactivity issues with FAM234A Antibody, Biotin conjugated?

Addressing potential cross-reactivity issues with FAM234A Antibody, Biotin conjugated requires systematic analytical and experimental approaches. First, researchers should thoroughly examine the antibody's documentation regarding the immunogen used (typically recombinant Human Protein FAM234A protein, amino acids 115-251, or specific peptide sequences) . Perform in silico analysis using protein sequence databases to identify proteins with sequence similarity to the immunogenic region of FAM234A, particularly focusing on the ITFG3 family members or proteins with similar structural motifs. Experimentally, conduct western blot analysis with both FAM234A-positive and negative control samples, looking for unexpected bands that might indicate cross-reactivity. To specifically address cross-reactivity concerns, perform peptide competition assays using both the specific immunizing peptide and structurally similar peptides from potential cross-reactive proteins. Additionally, validate antibody specificity using cells or tissues with FAM234A knockdown or knockout compared to wild-type controls. For complex samples, consider pre-adsorption of the antibody with proteins or cell lysates from species or tissues known to lack FAM234A expression to remove potentially cross-reactive antibody populations. When interpreting results, always include appropriate negative controls and, where possible, confirm key findings with alternative FAM234A antibodies that recognize different epitopes . These comprehensive approaches help ensure that observed signals are specifically attributable to FAM234A rather than cross-reactive proteins.

How can FAM234A Antibody, Biotin conjugated be integrated into multiplexed protein detection systems?

Integrating FAM234A Antibody, Biotin conjugated into multiplexed protein detection systems requires strategic planning to leverage biotin-streptavidin interactions while avoiding cross-detection issues. For fluorescence-based multiplexing, researchers can combine biotinylated FAM234A antibody with streptavidin conjugated to spectrally distinct fluorophores (e.g., Alexa Fluor 488, 555, or 647) that are compatible with available imaging systems . This approach works effectively when sequential detection protocols are employed, where complete detection of FAM234A is performed before introducing additional primary antibodies for other targets. For more sophisticated multiplexing, consider tyramide signal amplification (TSA) systems that utilize the biotinylated antibody with HRP-streptavidin conjugates to deposit biotin-tyramide at detection sites, followed by visualization with fluorophore-conjugated streptavidin . This method allows for antibody stripping and sequential detection of multiple proteins on the same sample. In microarray formats, the biotinylated FAM234A antibody can be spotted alongside other detection antibodies to create protein interaction networks when probed with complex biological samples. For mass cytometry applications, biotinylated antibodies can be detected using metal-conjugated streptavidin, enabling integration into CyTOF workflows for single-cell protein profiling. When designing multiplexed systems, researchers must carefully validate each component individually before combination and implement appropriate controls to assess potential cross-talk between detection channels . The exceptionally high affinity of biotin-streptavidin binding (Kd ≈ 10^-15 M) makes it particularly valuable for stable multiplex detection systems with minimal dissociation during sequential staining procedures.

What are the considerations for using FAM234A Antibody, Biotin conjugated in co-localization studies with confocal microscopy?

When conducting co-localization studies using FAM234A Antibody, Biotin conjugated with confocal microscopy, researchers must address several critical considerations. First, select an appropriate streptavidin conjugate with a fluorophore that has minimal spectral overlap with other fluorophores used in the experiment. Common choices include streptavidin-Alexa Fluor 488, 555, or 647, depending on the microscope's laser configuration and other fluorescent markers being used . Second, optimize the protocol to minimize background autofluorescence by employing Sudan Black B (0.1-0.3%) or copper sulfate (10mM) treatment, particularly for tissues with high endogenous fluorescence. Third, address potential endogenous biotin interference by pre-blocking with avidin/biotin blocking kits before introducing the biotinylated antibody . Fourth, establish proper controls including single-label controls to assess bleed-through and secondary-only controls to evaluate non-specific binding. Fifth, for detecting low-abundance FAM234A, implement signal amplification using tyramide amplification systems specifically designed for biotinylated antibodies, such as the Biotin XX Tyramide SuperBoost Kit . For accurate co-localization analysis, ensure proper alignment of detection channels and use appropriate quantitative co-localization metrics such as Pearson's correlation coefficient, Manders' overlap coefficient, or object-based co-localization analysis. During image acquisition, maintain consistent parameters (laser power, gain, offset) between samples and controls. For optimal resolution in subcellular co-localization, adapt the protocol for super-resolution microscopy techniques such as STED or STORM using appropriate streptavidin conjugates compatible with these advanced imaging modalities .

How can FAM234A Antibody, Biotin conjugated be utilized in chromatin immunoprecipitation (ChIP) experiments?

Adapting FAM234A Antibody, Biotin conjugated for chromatin immunoprecipitation (ChIP) experiments requires methodological modifications to the standard ChIP protocol. First, establish whether FAM234A has known or predicted DNA-binding capabilities or associates with chromatin-modifying complexes, as this information will guide experimental design and interpretation. For the ChIP procedure, begin with standard chromatin preparation through formaldehyde cross-linking of protein-DNA complexes, followed by chromatin fragmentation to 200-500 bp fragments via sonication or enzymatic digestion. When using the biotinylated FAM234A antibody for immunoprecipitation, two strategic approaches can be employed: direct or indirect capture. For direct capture, pre-incubate the biotinylated antibody with the chromatin sample to form antibody-protein-DNA complexes, then add streptavidin-coated magnetic beads for efficient complex isolation . This approach leverages the high-affinity biotin-streptavidin interaction (Kd ≈ 10^-15 M) for stringent purification. Alternatively, for indirect capture, use protein G magnetic beads coated with anti-biotin antibodies to precipitate the biotinylated FAM234A antibody complexes. Following either approach, implement stringent washing steps to remove non-specific interactions while preserving specific antibody-protein-DNA complexes. After reverse cross-linking and DNA purification, analyze the immunoprecipitated DNA using qPCR, sequencing (ChIP-seq), or other applicable methods to identify genomic regions associated with FAM234A. Throughout the process, include appropriate controls such as IgG control, input chromatin, and known positive/negative genomic regions to validate specificity of the findings .

How does FAM234A Antibody, Biotin conjugated compare to other FAM234A antibody conjugates?

FAM234A antibodies are available with several different conjugations, each offering distinct advantages for specific applications. The biotin-conjugated FAM234A antibody provides superior signal amplification capabilities compared to directly labeled conjugates, making it particularly valuable for detecting low-abundance targets . When compared to HRP-conjugated FAM234A antibodies, the biotin conjugate offers greater flexibility in detection systems, as it can be paired with various streptavidin-reporter conjugates based on experimental needs . The FITC-conjugated version provides direct fluorescence detection without requiring secondary reagents, which can be advantageous for reducing background in some applications, but typically offers lower sensitivity than the biotin-streptavidin system . The following table summarizes the comparative analysis of different FAM234A antibody conjugates:

For researchers needing maximum sensitivity and detection flexibility, the biotin-conjugated FAM234A antibody typically represents the optimal choice, particularly for ELISA applications where signal amplification can significantly improve detection limits .

What criteria should researchers consider when selecting between different suppliers of FAM234A Antibody, Biotin conjugated?

When selecting between different suppliers of FAM234A Antibody, Biotin conjugated, researchers should evaluate several critical criteria to ensure experimental success. First, examine immunogen information - antibodies generated against full recombinant proteins (such as recombinant Human Protein FAM234A protein, amino acids 115-251AA) may offer broader epitope recognition than those raised against synthetic peptides . Second, compare validation data provided by manufacturers, including western blot images, ELISA performance metrics, and positive control recommendations (e.g., MCF7 cell lysate) . Third, assess purification methods - antibodies purified by Protein G chromatography to >95% purity generally offer superior specificity compared to less stringently purified products . Fourth, evaluate conjugation quality through parameters like biotin-to-protein ratio, which affects both specificity and signal amplification potential. Fifth, consider tested applications - while all reviewed suppliers validate their FAM234A biotinylated antibodies for ELISA, some may offer additional validated applications like western blotting . Sixth, compare technical specifications such as concentration (typically 0.5-1.0 mg/ml), buffer composition (most contain glycerol, PBS, and preservatives like Proclin 300), and storage recommendations . Seventh, review host species and clonality - all identified suppliers offer rabbit polyclonal antibodies, which provide good sensitivity but may have lot-to-lot variation compared to monoclonals. Finally, consider manufacturer support, including availability of technical data sheets, application protocols, and responsive technical service for troubleshooting assistance. These comprehensive selection criteria help ensure experimental success with FAM234A Antibody, Biotin conjugated.

How do different detection systems impact the performance of FAM234A Antibody, Biotin conjugated?

The choice of detection system significantly impacts the performance of FAM234A Antibody, Biotin conjugated across various applications. Streptavidin-HRP systems provide excellent sensitivity for colorimetric or chemiluminescent detection in western blotting and ELISA, with detection limits typically in the low picogram range for optimized systems . These systems are ideal when quantitative measurement is the primary goal. Fluorescent detection using streptavidin conjugated to fluorophores (such as Alexa Fluor dyes) offers superior spatial resolution for microscopy applications while maintaining high sensitivity . The table below compares key performance metrics across different detection systems:

For signal amplification beyond standard streptavidin systems, tyramide signal amplification (TSA) technologies such as the Tyramide SuperBoost Kits can enhance FAM234A detection by 10-100 fold compared to conventional methods . This approach is particularly valuable for detecting low-abundance FAM234A in complex samples. For multiplex detection, researchers should consider spectrally distinct streptavidin conjugates that can be combined with other detection systems, noting that sequential detection protocols may be necessary to avoid cross-reactivity issues . When selecting a detection system, researchers should consider not only sensitivity requirements but also compatibility with available instrumentation, experimental timeline, and downstream analysis needs.