FAM234A Antibody, HRP conjugated

What is FAM234A and what cellular functions is it associated with?

FAM234A, also known as Protein ITFG3 or C16orf9 ITFG3, is a human protein with UniProt ID Q9H0X4. It has been cataloged in several biological databases including HGNC (ID: 14163), KEGG (hsa:83986), and STRING (9606.ENSP00000301678) . While the search results don't elaborate on its specific cellular functions, researchers typically use antibodies against FAM234A to investigate its expression patterns and interactions in human tissues and cell lines. The protein is encoded by a gene located on chromosome 16 and has been the subject of investigation in various cellular biology studies.

What applications is FAM234A Antibody, HRP conjugated suitable for?

FAM234A Antibody, HRP conjugated is primarily recommended for ELISA applications, as indicated in product listings from multiple suppliers . The HRP (horseradish peroxidase) conjugation makes it particularly suitable for detection systems that utilize chromogenic or chemiluminescent substrates. While the non-conjugated version of the antibody may be used for immunofluorescence (IF) at dilutions of 1:50-1:200, the HRP-conjugated version is specifically optimized for ELISA-based detection systems .

What is the difference between conjugated and non-conjugated FAM234A antibodies?

Non-conjugated FAM234A antibodies (such as CSB-PA887949LA01HU) require a secondary detection system, while HRP-conjugated versions (CSB-PA887949LB01HU) have the enzyme directly attached to the antibody molecule . This direct conjugation eliminates the need for secondary antibodies, reducing background and potential cross-reactivity issues in assays. The conjugation process involves the directional covalent bonding of HRP to the antibody molecule, which allows for one-step detection in immunoassays while maintaining the antibody's specificity for the FAM234A protein .

How should FAM234A Antibody, HRP conjugated be stored for optimal performance?

For optimal stability and performance, FAM234A Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . It's important to avoid repeated freeze-thaw cycles as these can degrade both the antibody and the HRP enzyme activity. The antibody is typically supplied in a buffer containing preservatives such as Proclin 300 (0.03%) and stabilizers like glycerol (50%), formulated in a PBS buffer at pH 7.4 . These components help maintain the antibody's stability and functional properties during storage.

What are the recommended working dilutions for FAM234A Antibody, HRP conjugated?

For ELISA applications, which is the primary recommended use for FAM234A Antibody, HRP conjugated, specific working dilutions should be determined experimentally for optimal results . While exact dilutions for the HRP-conjugated version aren't specified in the search results, the non-conjugated version is recommended at dilutions of 1:50-1:200 for immunofluorescence (IF) applications . Researchers should perform titration experiments to determine the optimal concentration that provides the best signal-to-noise ratio for their specific experimental conditions and sample types.

What buffer conditions are optimal when using FAM234A Antibody, HRP conjugated?

When working with HRP-conjugated antibodies including FAM234A, it's crucial to avoid buffer components that can interfere with enzyme activity. Specifically, sodium azide should be strictly avoided as it is an irreversible inhibitor of HRP and will compromise assay performance . The antibody is typically supplied in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 . For dilution and assay buffers, 10-50mM amine-free buffers (such as HEPES, MES, MOPS, or phosphate) with pH range 6.5-8.5 are recommended. Moderate concentrations of Tris buffer (<20mM) may be tolerated, but buffers containing nucleophilic components such as primary amines and thiols should be avoided as they may interfere with antibody function .

What are the expected molecular weights for FAM234A detection?

For accurate interpretation of results when using FAM234A Antibody, HRP conjugated, researchers should be aware that the human FAM234A protein has a predicted molecular weight consistent with its UniProt entry (Q9H0X4) . The antibody is raised against a specific region of the human FAM234A protein (amino acids 115-251) , which should be considered when interpreting experimental results, especially in Western blot applications. Post-translational modifications may result in migration patterns that differ from the predicted molecular weight, so proper positive controls are essential for accurate identification.

How can I validate the specificity of FAM234A Antibody, HRP conjugated?

To validate the specificity of FAM234A Antibody, HRP conjugated, researchers should implement multiple control strategies. These include using known positive samples expressing FAM234A, negative controls where the protein is absent or knocked down, pre-absorption controls using the immunizing peptide (amino acids 115-251 of human FAM234A protein) , and comparison with other antibodies targeting different epitopes of the same protein. For polyclonal antibodies like this one, batch-to-batch variation should be considered, and new lots should be validated against previously characterized lots to ensure consistent specificity and sensitivity.

What are the considerations for using FAM234A Antibody, HRP conjugated in multiplex assays?

When incorporating FAM234A Antibody, HRP conjugated into multiplex assays, researchers must consider several factors. The HRP conjugate produces a colorimetric or chemiluminescent signal that may limit multiplex capabilities compared to fluorescent conjugates. If designing a multiplex ELISA, it's important to select other detection enzymes with distinct substrates that won't cross-react. Alternatively, sequential detection protocols may be implemented, where the HRP signal is developed and captured before introducing additional detection antibodies. The polyclonal nature of the antibody (raised in rabbit) should also be considered when selecting other antibodies for multiplexing to avoid species cross-reactivity.

What are the critical parameters affecting the performance of FAM234A Antibody, HRP conjugated in ELISA?

Several critical parameters influence the performance of FAM234A Antibody, HRP conjugated in ELISA applications. These include coating concentration of capture antibodies (if using a sandwich ELISA format), blocking efficiency to prevent non-specific binding, antibody concentration (optimal working dilution should be determined experimentally), sample preparation methods, incubation times and temperatures, washing stringency, substrate selection, and detection timing. The purity of the antibody preparation is also important - the FAM234A antibody from some suppliers is Protein G purified with >95% purity , which can impact background levels and signal specificity.

How can I optimize signal-to-noise ratio when using FAM234A Antibody, HRP conjugated?

To optimize signal-to-noise ratio with FAM234A Antibody, HRP conjugated, implement these strategies: (1) Optimize antibody concentration through careful titration experiments; (2) Ensure thorough blocking with appropriate blocking reagents compatible with HRP detection systems; (3) Include 0.05-0.1% Tween-20 in wash buffers to reduce non-specific binding; (4) Extend washing steps to thoroughly remove unbound antibody; (5) Consider sample pre-clearing to remove components that might contribute to background; (6) Select an appropriate substrate with sensitivity matched to your expected signal level; and (7) Optimize incubation times for both antibody binding and substrate development phases of your assay.

What controls should be included when using FAM234A Antibody, HRP conjugated?

A comprehensive control strategy for experiments using FAM234A Antibody, HRP conjugated should include: (1) Positive controls - samples known to express FAM234A; (2) Negative controls - samples known to lack FAM234A expression; (3) Secondary antibody-only controls (if using indirect detection methods); (4) Substrate-only controls to assess background from the detection system; (5) Isotype controls using irrelevant rabbit IgG at the same concentration ; (6) Blocking peptide controls using the immunogen peptide (amino acids 115-251 of human FAM234A) ; and (7) Serial dilution controls of positive samples to confirm signal linearity. These controls help validate assay specificity, sensitivity, and reproducibility.

How can I quantify the expression levels of FAM234A using HRP conjugated antibodies?

To quantify FAM234A expression levels using HRP-conjugated antibodies, establish a standard curve using recombinant FAM234A protein at known concentrations. For ELISA applications, this enables absolute quantification through regression analysis of the standard curve. Alternatively, relative quantification can be performed by normalizing signal intensity to total protein concentration or housekeeping proteins. When comparing expression across different samples or conditions, include consistent positive controls across all experiments to account for inter-assay variation. For more precise quantification, consider a sandwich ELISA format using a capture antibody targeting a different epitope of FAM234A than the HRP-conjugated detection antibody.

How should I prepare samples for optimal detection of FAM234A using HRP conjugated antibodies?

Sample preparation is crucial for successful detection of FAM234A. For cell or tissue lysates, use a lysis buffer compatible with HRP activity (avoid sodium azide) . Including protease inhibitors is essential to prevent degradation of FAM234A. For ELISA applications, samples may require dilution in appropriate assay buffers (10-50mM amine-free buffers such as HEPES, MES, MOPS, or phosphate with pH range 6.5-8.5) . Pre-clearing samples by centrifugation or filtration can remove particulates that might interfere with antibody binding or cause high background. Additionally, optimization of protein concentration is important—too concentrated samples may lead to hook effects, while too dilute samples may yield signals below detection limits.

Can FAM234A Antibody, HRP conjugated be used for immunohistochemistry applications?

While the search results specifically mention ELISA as the recommended application for FAM234A Antibody, HRP conjugated , HRP-conjugated antibodies are generally suitable for immunohistochemistry (IHC) applications. For IHC, sample preparation would include appropriate fixation (typically formalin or paraformaldehyde), antigen retrieval optimization (heat-induced or enzymatic), and endogenous peroxidase blocking to prevent non-specific signal. Since the non-conjugated version is recommended for immunofluorescence at dilutions of 1:50-1:200 , similar dilution ranges might serve as a starting point for IHC optimization with the HRP-conjugated version, though specific validation would be required.

How should I troubleshoot non-specific binding when using FAM234A Antibody, HRP conjugated?

If experiencing non-specific binding with FAM234A Antibody, HRP conjugated, implement these troubleshooting steps: (1) Increase blocking stringency using different blocking agents (BSA, casein, or commercial blocking solutions); (2) Optimize antibody dilution—try using more dilute antibody solutions; (3) Add 0.1-0.5% detergent (Tween-20 or Triton X-100) to reduce hydrophobic interactions; (4) Increase washing duration and number of wash steps; (5) Pre-absorb the antibody with proteins from the species being tested if cross-reactivity is suspected; (6) Use more stringent buffer conditions during antibody incubation; and (7) Consider alternative blocking agents if standard protocols are ineffective. Remember that polyclonal antibodies like this one may have batch-to-batch variation in non-specific binding characteristics .

What is the stability of FAM234A Antibody, HRP conjugated under different experimental conditions?

The stability of FAM234A Antibody, HRP conjugated is influenced by several factors. The antibody is supplied in a buffer containing 50% glycerol and 0.03% Proclin 300 as a preservative , which helps maintain stability during storage. HRP conjugates generally maintain activity for at least 12 months when stored properly at -20°C or -80°C and protected from repeated freeze-thaw cycles . During experiments, HRP activity is temperature-sensitive and optimal at room temperature or 37°C depending on the specific assay. Extended incubations at elevated temperatures may reduce activity. The enzyme is also pH-sensitive, with optimal activity between pH 6.0-7.0. Exposure to strong oxidizing or reducing agents, sodium azide, and certain metal ions should be avoided as these can irreversibly inactivate HRP .

How do different detection substrates affect the sensitivity when using FAM234A Antibody, HRP conjugated?

The choice of substrate significantly impacts the sensitivity achieved with FAM234A Antibody, HRP conjugated. Chromogenic substrates like TMB (3,3',5,5'-Tetramethylbenzidine) provide visual color development suitable for basic ELISA applications but offer limited sensitivity. Enhanced chemiluminescent (ECL) substrates provide substantially higher sensitivity, with signal amplification enabling detection of lower protein concentrations. Super-enhanced chemiluminescent substrates can further improve sensitivity by 10-100 fold compared to standard ECL. Chemifluorescent substrates offer another high-sensitivity option with the additional benefit of signal stability over time. The optimal substrate selection depends on the expected abundance of FAM234A in your samples, the detection equipment available, and whether quantitative analysis is required.

What are the considerations for using FAM234A Antibody, HRP conjugated in high-throughput screening?

For high-throughput screening applications using FAM234A Antibody, HRP conjugated, researchers should consider: (1) Assay miniaturization to conserve antibody and reduce costs; (2) Automation compatibility of all reagents and protocols; (3) Signal stability over time to accommodate batch processing; (4) Lot-to-lot consistency of the antibody, with large-scale studies ideally using a single lot; (5) Robust positive and negative controls on each plate to normalize for plate-to-plate variation; (6) Optimized washing protocols that balance thoroughness with efficiency; (7) Selection of detection substrates with appropriate signal windows and stability; and (8) Statistical methods for data normalization and hit identification appropriate for the specific experimental design.