NFKBIL1 Antibody, HRP conjugated

What is NFKBIL1 and why is it significant in immunological research?

NFKBIL1 (Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells Inhibitor-Like 1) is a divergent member of the I-kappa-B family of proteins. Its complete function remains under investigation, but it has significant research interest due to its genomic location within the major histocompatibility complex (MHC) class I region on chromosome 6 (6p21.33). The gene encodes a protein that may play a role in immune regulation through NF-kB pathway modulation . Research interest in NFKBIL1 has increased due to its associations with inflammatory conditions including inflammatory bowel disease, cardiac manifestations of neonatal lupus, and rheumatoid arthritis . When designing experiments targeting this protein, researchers should consider its potential regulatory functions in immune responses and inflammation.

What are the key specifications of commercially available NFKBIL1 antibodies with HRP conjugation?

NFKBIL1 antibodies with Horseradish Peroxidase (HRP) conjugation are typically rabbit polyclonal antibodies that target specific epitopes of the human NFKBIL1 protein. These antibodies are produced against recombinant fragments or synthetic peptides derived from human NFKBIL1. Available products feature different target regions, including internal epitopes and specific amino acid regions such as AA 185-312 . Most commercially available options are rabbit-derived polyclonal antibodies with IgG isotype . The HRP conjugation provides direct enzymatic labeling, eliminating the need for secondary antibody incubation steps in certain applications. Buffer compositions typically include preservatives like 0.03% Proclin 300, 50% Glycerol, and PBS at pH 7.4 . These specifications should guide researchers in selecting the appropriate antibody for their experimental design.

What is the optimal storage and handling protocol to maintain antibody activity?

To maintain optimal activity of NFKBIL1 antibody with HRP conjugation, consistent storage at -20°C or -80°C is recommended . Repeated freeze-thaw cycles significantly reduce antibody performance, so aliquoting upon receipt is advisable for long-term experimental planning. When working with the antibody, minimize exposure to room temperature and promptly return to cold storage after use. The buffer composition containing 50% glycerol helps prevent complete freezing and preserves antibody integrity . For applications requiring diluted working solutions, prepare them fresh and use immediately for optimal signal intensity. Additionally, exposure to strong light should be minimized to prevent photobleaching of the HRP moiety. Following proper handling protocols ensures consistent experimental results and extends the usable life of the antibody reagent.

What are the validated experimental applications for NFKBIL1 antibody with HRP conjugation?

NFKBIL1 antibody with HRP conjugation has been validated for several experimental applications, with ELISA (Enzyme-Linked Immunosorbent Assay) being the primary recommended application with a typical dilution of 1:1000 . Western blotting is also supported at working dilutions ranging from 1:100-500, allowing for protein detection in cell and tissue lysates . Additional applications include flow cytometry and immunofluorescence, typically used at dilutions of 1:10-50 . The HRP conjugation provides direct enzymatic detection capability, simplifying experimental workflows by eliminating the need for secondary antibody steps. When designing experiments, researchers should conduct preliminary titration studies to determine optimal antibody concentration for their specific sample types and detection systems, as sensitivity can vary between different detection methods and instrumentation setups.

How should researchers optimize Western blot protocols when using HRP-conjugated NFKBIL1 antibody?

When optimizing Western blot protocols with HRP-conjugated NFKBIL1 antibody, researchers should first determine appropriate protein loading (25-50μg total protein) and separation conditions, considering the predicted molecular weight of NFKBIL1. Following protein transfer to PVDF membrane (recommended over nitrocellulose for this application), membrane blocking should be optimized using 5% non-fat milk or 3-5% BSA in TBST. The HRP-conjugated NFKBIL1 antibody works effectively at dilutions between 1:100-500 in blocking buffer . Incubation time and temperature (typically overnight at 4°C or 2 hours at room temperature) should be empirically determined. After thorough washing with TBST buffer (4-5 washes of 5 minutes each), direct chemiluminescent detection can be performed without secondary antibody. Controls should include positive control lysates such as Jurkat or Raji cell lines, which have demonstrated specific band detection at approximately 50 kDa under reducing conditions . For troubleshooting weak signals, extended exposure times or enhanced chemiluminescent substrates may improve detection sensitivity.

What methodological considerations are important when using NFKBIL1 antibody in ELISA applications?

For optimal ELISA performance with HRP-conjugated NFKBIL1 antibody, researchers should implement a systematic optimization strategy. Begin with antibody titration at dilutions ranging from 1:500 to 1:2000, with 1:1000 serving as the recommended starting point . Coating concentration of capture antigen should be determined experimentally (typically 1-10 μg/ml), along with optimized blocking buffer composition (3-5% BSA or non-fat milk in PBS). Incubation times and temperatures significantly impact sensitivity and specificity—standard protocols suggest 1-2 hours at room temperature or overnight at 4°C for antibody binding steps. Multiple wash steps (4-5 times) using PBS-Tween (0.05%) between each reagent addition are crucial for reducing background. For detection, TMB substrate is commonly used with HRP-conjugated antibodies, with reaction time carefully monitored to avoid oversaturation. Standard curves should be prepared using recombinant NFKBIL1 protein for quantitative analysis. When comparing results across experimental conditions, maintaining consistency in all protocol parameters is essential for reliable data interpretation.

What are common troubleshooting strategies for false negative or weak signals with NFKBIL1 antibody?

When encountering false negative or weak signals with NFKBIL1 antibody with HRP conjugation, researchers should systematically evaluate multiple factors. First, verify target protein expression in the sample type—NFKBIL1 expression may vary significantly between tissue and cell types. Antibody concentration should be increased incrementally from the recommended 1:1000 dilution for ELISA or 1:100-500 for Western blotting . Check antibody viability by testing activity using positive control samples where NFKBIL1 expression is confirmed. Detection system sensitivity may need enhancement through extended substrate incubation or using high-sensitivity chemiluminescent reagents. If the antibody targets the C-terminal region (as with many NFKBIL1 antibodies) , protein modifications or truncations affecting this epitope could prevent binding. Sample preparation methods should be evaluated, as overly harsh conditions may denature the target epitope. For Western blotting specifically, insufficient protein transfer or inappropriate blocking reagents could interfere with antibody accessibility. Implementing these systematic troubleshooting approaches will help identify and resolve causes of weak or absent signals.

How can researchers verify the specificity of NFKBIL1 antibody reactions in their experimental systems?

Verifying NFKBIL1 antibody specificity requires implementing multiple complementary validation approaches. First, researchers should utilize positive and negative control samples—cell lines with confirmed high NFKBIL1 expression (such as Jurkat or Raji human cell lines) versus those with low or no expression . A critical validation approach is peptide competition assay, where pre-incubation of the antibody with excess immunizing peptide should significantly reduce or eliminate specific signal. For definitive specificity confirmation, NFKBIL1 knockdown or knockout experiments using siRNA or CRISPR-Cas9 should demonstrate corresponding signal reduction. Western blotting should reveal a specific band at the expected molecular weight (approximately 50 kDa) without non-specific bands. Cross-reactivity assessment is important when working with non-human samples, though many NFKBIL1 antibodies show broad species reactivity due to high sequence conservation (100% identity across multiple species including human, mouse, dog, bovine, and goat) . Implementing multiple validation methods provides stronger evidence for antibody specificity than any single approach alone.

What quality control tests should researchers perform when receiving a new lot of NFKBIL1 antibody?

Upon receiving a new lot of NFKBIL1 antibody with HRP conjugation, researchers should implement a comprehensive quality control testing regimen before incorporating it into ongoing experiments. Begin with physical inspection for visible precipitation or discoloration that might indicate degradation. Protein concentration verification using absorbance measurement at 280nm provides basic confirmation of antibody content. Functional validation should include side-by-side comparison with previously validated lots in standardized assays like Western blot or ELISA using consistent positive control samples. For Western blotting, verify correct molecular weight detection (~50 kDa) and similar signal intensity compared to previous lots under identical conditions. For ELISA applications, compare standard curves and EC50 values between lots to ensure equivalent sensitivity. Specificity should be confirmed through peptide competition assays. Additionally, lot-to-lot variations in optimal working dilutions should be established through titration experiments. Documentation of all validation results in laboratory records enables tracking of antibody performance over time and across different experimental applications.

What is known about NFKBIL1's involvement in disease pathogenesis and how might this inform research applications?

NFKBIL1 has been implicated in several immune-mediated disease processes through genomic association studies. Research evidence links NFKBIL1 genetic variants to inflammatory bowel disease pathogenesis, suggesting its role in host-microbe interactions that influence intestinal inflammation . Additionally, genome-wide association studies have identified NFKBIL1 as a candidate locus (6p21) associated with cardiac manifestations of neonatal lupus, indicating potential involvement in autoimmune-mediated cardiac inflammation and damage . The strongest disease association appears to be with rheumatoid arthritis, where NFKBIL1 variants may influence disease susceptibility or progression . The gene's location within the MHC class I region on chromosome 6 (6p21.33) places it in a genomic neighborhood dense with immune regulatory genes, suggesting functional relationships with the broader immunological framework . When designing research using NFKBIL1 antibodies, these disease associations suggest value in examining NFKBIL1 expression or modification in inflammatory microenvironments, autoimmune contexts, and in response to various immune stimuli. Comparative studies examining NFKBIL1 in affected versus healthy tissues may provide insights into its role in disease mechanisms.

How might post-translational modifications of NFKBIL1 affect antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) of NFKBIL1 could significantly impact antibody recognition based on epitope accessibility and modification state. While specific PTM data for NFKBIL1 is limited in the provided search results, researchers should consider that as a member of the I-kappa-B family of proteins, phosphorylation and ubiquitination may play regulatory roles similar to other family members. Antibodies targeting specific regions, particularly internal epitopes or C-terminal domains , may be differentially affected by PTMs occurring within or adjacent to these recognition sites. When analyzing conflicting experimental results or unexpected antibody behavior, researchers should consider the possibility that cellular activation states or experimental conditions might alter NFKBIL1 modification patterns. For experiments specifically investigating NFKBIL1 regulation, comparing results from antibodies recognizing distinct epitopes may reveal condition-dependent epitope masking. Additionally, sample preparation methods that preserve or disrupt specific modifications (such as phosphatase inhibitors in lysis buffers) could affect detection outcomes. To address these concerns, researchers can employ multiple antibodies targeting different NFKBIL1 epitopes and compare detection patterns across various experimental conditions and cellular activation states.

What detection systems are optimal for visualizing HRP-conjugated NFKBIL1 antibody binding?

For optimal visualization of HRP-conjugated NFKBIL1 antibody binding, researchers should select detection systems based on their specific application requirements for sensitivity, dynamic range, and signal persistence. In Western blotting applications, enhanced chemiluminescence (ECL) detection provides excellent sensitivity with low background when working with recommended antibody dilutions (1:100-500) . For quantitative Western analyses, digital imaging systems with extended dynamic range capabilities are preferable to film-based detection. In ELISA applications, colorimetric substrates like TMB (3,3',5,5'-tetramethylbenzidine) offer cost-effective detection with appropriate sensitivity for most applications at the recommended 1:1000 dilution . For higher sensitivity requirements, chemiluminescent ELISA substrates can enhance detection limits by approximately 10-100 fold. In immunohistochemistry applications, DAB (3,3'-diaminobenzidine) provides a stable, permanent reaction product ideal for long-term slide storage and analysis. Fluorescent tyramide signal amplification (TSA) systems compatible with HRP can dramatically increase sensitivity for applications with limited target abundance while maintaining compatibility with multicolor fluorescence workflows. Selection of the optimal detection system should balance sensitivity requirements with practical considerations including equipment availability, signal stability needs, and quantification objectives.

How might the HRP conjugation affect antibody stability and shelf-life, and what measures can extend usability?

The HRP conjugation to NFKBIL1 antibody introduces specific stability considerations beyond those of unconjugated antibodies. The enzymatic HRP moiety is more susceptible to denaturation and oxidative damage than the antibody portion, potentially limiting shelf-life. To maximize stability, storage at -20°C or -80°C is strongly recommended, with -80°C providing superior long-term preservation for periods exceeding 6 months . Upon receipt, antibody aliquoting into single-use volumes prevents damaging freeze-thaw cycles that particularly affect conjugated antibodies. The standard storage buffer containing 50% glycerol serves as a cryoprotectant, preventing complete freezing and associated denaturation damage . Researchers should strictly avoid sodium azide in any working solutions, as it irreversibly inhibits HRP activity even at low concentrations. Exposure to strong oxidizing agents and extreme pH conditions should be minimized. For working solutions, preparation with stabilizing proteins (0.1-0.5% BSA) can extend short-term stability. Light exposure should be limited as it may accelerate HRP degradation. When stored and handled properly, HRP-conjugated antibodies typically maintain activity for 12-18 months, though gradual sensitivity reduction may occur. Regular performance testing using standardized positive controls allows researchers to monitor activity over time and adjust protocols accordingly.