UIMC1 Antibody, HRP conjugated

What is UIMC1 and what is its biological function?

UIMC1 (Ubiquitin Interaction Motif Containing 1) is a nuclear protein that functions as a ubiquitin-binding protein, specifically recognizing and binding 'Lys-63'-linked ubiquitin. It plays a central role in the BRCA1-A complex by specifically binding 'Lys-63'-linked ubiquitinated histones H2A and H2AX at DNA lesion sites, leading to the recruitment of the BRCA1-BARD1 heterodimer to sites of DNA damage at double-strand breaks (DSBs) . The BRCA1-A complex also possesses deubiquitinase activity that specifically removes 'Lys-63'-linked ubiquitin on histones H2A and H2AX . Additionally, UIMC1 may indirectly act as a transcriptional repressor by inhibiting the interaction of NR6A1 with the corepressor NCOR1 .

UIMC1 is also known by several synonyms including BRCA1-A complex subunit RAP80, Receptor-associated protein 80, Retinoid X receptor-interacting protein 110, and RAP80 RXRIP110 .

What is an HRP-conjugated antibody and how does it function?

Horseradish Peroxidase (HRP)-conjugated antibodies are immunoglobulins that have been chemically linked to the enzyme HRP. This conjugation enables sensitive detection in various immunoassays through the catalytic properties of HRP, which can convert chromogenic, chemiluminescent, or fluorogenic substrates into detectable signals.

The conjugation process typically utilizes the glycoprotein nature of HRP, whose polysaccharide chains serve as attachment points for cross-linking reactions . Two primary methods for conjugation include:

• Reductive amination: Mild oxidation of sugar moieties on HRP with sodium periodate generates reactive aldehyde groups that can form covalent bonds with amine groups on antibodies in the presence of sodium cyanoborohydride .

• Heterobifunctional crosslinkers: Water-soluble reagents such as sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) and N-succinimidyl S-acetylthioacetate (SATA) can be used to generate stable antibody-HRP conjugates .

The resulting HRP-conjugated antibody maintains both the specific binding capacity of the antibody and the enzymatic activity of HRP, making it particularly useful for detection in ELISA and other immunoassays.

What are the primary applications of UIMC1 Antibody, HRP conjugated?

The UIMC1 Antibody, HRP conjugated, is primarily optimized for use in Enzyme-Linked Immunosorbent Assay (ELISA) . This application leverages the specific binding of the antibody to UIMC1 protein and the signal amplification capabilities of the HRP conjugate to achieve sensitive detection.

While the HRP-conjugated version is specifically tested for ELISA applications, other forms of UIMC1 antibodies have demonstrated utility in multiple applications including:

Researchers should note that while the HRP-conjugated UIMC1 antibody is primarily validated for ELISA, experimental validation may be required to determine its efficacy in other applications where direct detection is beneficial.

What are the optimal storage and handling conditions for UIMC1 Antibody, HRP conjugated?

To maintain optimal activity and stability of UIMC1 Antibody, HRP conjugated, the following storage and handling conditions are recommended:

• Storage Temperature: Store at -20°C or -80°C upon receipt . Long-term storage at -20°C is stable for products containing 50% glycerol buffer systems .

• Buffer Composition: The antibody is typically supplied in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 .

• Aliquoting: For frequent use, it is advisable to prepare small aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce antibody activity .

• Working Conditions: When working with the antibody, maintain cold chain practices by keeping it on ice during experiments.

• Physical Form: The antibody is provided in liquid form, which facilitates handling while minimizing potential denaturation associated with lyophilization and reconstitution .

Adherence to these storage and handling guidelines will help ensure consistent performance and extend the shelf life of the UIMC1 Antibody, HRP conjugated.

What are the recommended dilutions for different applications of UIMC1 antibodies?

While specific dilution recommendations for UIMC1 Antibody, HRP conjugated for ELISA applications are not explicitly provided in the search results, researchers should conduct titration experiments to determine optimal concentrations. The following table presents recommended dilutions for various applications of non-HRP conjugated UIMC1 antibodies, which can serve as a starting reference:

It is crucial to note that optimal dilutions are sample-dependent and may vary based on the specific experimental design, detection method, and target abundance. For consistent and reliable results, it is strongly recommended that researchers titrate the antibody in their specific testing systems .

How does species reactivity differ among various UIMC1 antibodies?

The species reactivity of UIMC1 antibodies varies depending on the specific product and manufacturer. Understanding these differences is crucial for selecting the appropriate antibody for cross-species studies. The following table summarizes the reported species reactivity for different UIMC1 antibody products:

The variations in species reactivity are likely due to differences in the immunogen used for antibody production and the conservation of epitope sequences across species. For instance, the HRP-conjugated UIMC1 antibodies from both Assay Genie and Qtonics use a recombinant human BRCA1-A complex subunit RAP80 protein (amino acids 161-235) as the immunogen , which may account for their specific reactivity to human samples.

When planning experiments involving multiple species, researchers should carefully evaluate the validated reactivity of the available antibodies and consider performing their own cross-reactivity tests if needed.

What validation methods should be employed to verify UIMC1 antibody specificity?

Ensuring antibody specificity is critical for generating reliable experimental data. For UIMC1 antibodies, including HRP-conjugated versions, several validation methods should be considered:

• Western Blot Analysis: This is a primary validation method that confirms the antibody detects a protein of the expected molecular weight. For UIMC1, multiple molecular weight forms have been observed:

  • Calculated molecular weight: 80 kDa

  • Observed molecular weights: 97 kDa, 80 kDa, 71-75 kDa, 61 kDa or 120 kDa/100 kDa

  Western blot validation should include positive controls such as HeLa cells or mouse colon tissue, which have been shown to express detectable levels of UIMC1 .

• Cell and Tissue Panel Testing: Validation across multiple cell lines and tissues helps establish expression patterns. Positive detection has been reported in:

  • Cell lines: COLO 320, HeLa, Raji, MCF-7

  • Tissues: Human testis, mouse colon

• Knockout/Knockdown Controls: Testing the antibody in UIMC1 knockout or knockdown samples provides stringent specificity confirmation.

• Immunoprecipitation (IP) followed by Mass Spectrometry: This approach can verify that the antibody is capturing the intended target protein.

• ELISA with Recombinant Protein: For HRP-conjugated antibodies specifically designed for ELISA, validation should include detection of purified recombinant UIMC1 protein with appropriate concentration curves.

Rigorous validation using multiple methods ensures that experimental results reflect genuine UIMC1 biology rather than non-specific interactions.

How does the HRP conjugation affect the binding properties of UIMC1 antibodies?

The HRP conjugation process can potentially impact the binding properties of UIMC1 antibodies through several mechanisms. Understanding these effects is crucial for experimental design and interpretation:

• Steric Hindrance: The addition of the HRP enzyme (approximately 44 kDa) may introduce steric constraints that could affect antibody binding to certain epitopes, particularly if the conjugation occurs near the antigen-binding site. This may be particularly relevant for UIMC1, which is involved in complex protein-protein interactions within the BRCA1-A complex .

• Epitope Accessibility: HRP conjugation using methods that target lysine residues on the antibody (e.g., reductive amination with sodium periodate ) might modify amino groups in or near the antigen-binding region, potentially altering epitope recognition.

• Binding Kinetics: The conjugation can affect the on/off rates of antibody-antigen binding. This might manifest as changes in assay sensitivity or in the time required to reach equilibrium binding.

• Antibody Concentration Effects: The effective concentration of binding-competent antibody may be reduced after conjugation due to some molecules being inactivated during the conjugation process. This necessitates careful titration of HRP-conjugated UIMC1 antibodies in each experimental system.

For optimal experimental outcomes, researchers should compare the performance of HRP-conjugated UIMC1 antibodies with unconjugated versions in parallel assays when possible, particularly when establishing new assays or investigating novel UIMC1 interactions.

What are the optimal experimental conditions for detecting UIMC1 in DNA damage response studies?

When investigating UIMC1's role in DNA damage response pathways, several experimental conditions should be optimized to ensure robust and reproducible results:

• DNA Damage Induction: Since UIMC1 (RAP80) is recruited to DNA damage sites, treatment conditions should be optimized:

  • Ionizing radiation: Typically 2-10 Gy, with protein collection 0.5-4 hours post-irradiation

  • Chemical agents: Consider etoposide (10-100 μM), neocarzinostatin (100-500 ng/ml), or mitomycin C (0.5-2 μg/ml)

• Chromatin Fractionation: Because UIMC1 binds to ubiquitinated histones H2A and H2AX at DNA lesion sites , chromatin fractionation protocols should be optimized to retain these interactions:

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

  • Use deubiquitinase inhibitors (e.g., N-ethylmaleimide) to preserve ubiquitination status

• Co-immunoprecipitation Conditions: For studying UIMC1 interactions with the BRCA1-BARD1 heterodimer:

  • Buffer composition: Include 0.1-0.5% NP-40 or Triton X-100 to maintain protein complexes

  • Salt concentration: 120-150 mM NaCl is typically optimal for preserving nuclear protein complexes

• Immunofluorescence Detection:

  • Fixation: 4% paraformaldehyde for 10-15 minutes preserves nuclear architecture

  • Permeabilization: 0.2% Triton X-100 for 5-10 minutes allows antibody access

  • Antigen retrieval: For tissue sections, consider TE buffer pH
    9.0 or citrate buffer pH 6.0

• Western Blot Detection:

  • Lysis buffer: Include phosphatase inhibitors to preserve post-translational modifications

  • Gel percentage: 7.5-10% polyacrylamide gels provide optimal resolution for the 80-120 kDa range where UIMC1 is detected

Optimizing these conditions will enhance detection sensitivity and specificity when studying UIMC1's dynamic behavior in DNA damage response pathways.

How can researchers troubleshoot inconsistent results when using UIMC1 Antibody, HRP conjugated?

When encountering inconsistent results with UIMC1 Antibody, HRP conjugated, consider the following systematic troubleshooting approach:

• Antibody Integrity Issues:

  • Verify storage conditions: Improper storage or excessive freeze-thaw cycles can degrade HRP activity or antibody binding

  • Check expiration date: HRP enzyme activity diminishes over time

  • Solution: Prepare fresh working dilutions from minimally thawed stock aliquots

• Assay-Specific Considerations for ELISA:

  • Coating conditions: Optimize antigen concentration and buffer pH

  • Blocking efficiency: Insufficient blocking leads to high background, while excessive blocking may mask epitopes

  • Wash stringency: Inadequate washing increases background, while excessive washing may remove specific signal

  • Substrate handling: Ensure HRP substrate is fresh and protected from light

  • Solution: Perform checkerboard titrations of antibody and antigen concentrations

• Sample Preparation Concerns:

  • Protein denaturation: UIMC1 has multiple functional domains that may be sensitive to denaturation conditions

  • Post-translational modifications: DNA damage-induced modifications may affect antibody recognition

  • Solution: Try native conditions or different extraction methods

• Technical Considerations:

  • Detection system sensitivity: Ensure your detection system falls within the linear range for HRP signal

  • Temperature effects: Consistent temperature during incubation steps improves reproducibility

  • Solution: Standardize all protocol parameters and include robust positive and negative controls

• UIMC1-Specific Considerations:

  • Expression level variability: UIMC1 expression may change with cell cycle or stress conditions

  • Isoform detection: Alternative splicing results in multiple UIMC1 transcript variants

  • Solution: Characterize UIMC1 expression in your experimental system using multiple detection methods

Maintaining detailed experimental records and including appropriate controls will facilitate identifying the source of inconsistency and developing effective solutions.

What methodological considerations apply when integrating UIMC1 Antibody, HRP conjugated in multiplex assays?

Integrating UIMC1 Antibody, HRP conjugated into multiplex assays requires careful methodological considerations to ensure specificity, sensitivity, and compatibility with other detection systems:

• Antibody Cross-Reactivity Assessment:

  • Perform single-plex controls to establish baseline performance

  • Test for cross-reactivity with other primary and secondary antibodies in the multiplex panel

  • Validate specificity with appropriate blocking experiments

• Signal Separation Strategies:

  • Substrate selection: When combining with other HRP-conjugated antibodies, consider sequential detection with different substrates

  • For spectral multiplexing, use HRP substrates with distinct emission spectra (e.g., fluorescent HRP substrates)

  • Employ spatial separation techniques such as multi-well formats or compartmentalized assay systems

• Signal Amplification Calibration:

  • Determine the linear dynamic range of HRP signal for UIMC1 detection

  • Calibrate exposure times or substrate development periods to balance signals from targets with different abundance levels

  • Consider tyramide signal amplification (TSA) for enhanced sensitivity while maintaining multiplexing capability

• UIMC1-Specific Considerations:

  • Subcellular localization: UIMC1 is predominantly nuclear and associates with chromatin upon DNA damage

  • Protein interactions: UIMC1 forms complexes with BRCA1 and other DNA repair proteins

  • For multiplex imaging, optimize fixation and permeabilization conditions to preserve these interactions

• Data Analysis and Normalization:

  • Implement appropriate background subtraction methods for each detection channel

  • Use reference standards for inter-assay normalization

  • Consider computational approaches to deconvolve overlapping signals

By addressing these methodological considerations, researchers can successfully integrate UIMC1 Antibody, HRP conjugated into multiplex assays for comprehensive analysis of DNA damage response pathways and BRCA1-A complex dynamics.