INTS10 Antibody, Biotin conjugated

What is INTS10 and what is its biological significance?

INTS10 (Integrator Complex Subunit 10) is a critical component of the Integrator complex that associates with the C-terminal domain of RNA polymerase II large subunit (POLR2A) and mediates 3-prime end processing of small nuclear RNAs, including U1 . The protein has a calculated molecular weight of approximately 82 kDa, though it may appear between 70-82 kDa in experimental conditions . INTS10 is also known by alternative names including C8orf35 and INT10 . The integrator complex plays significant roles in transcriptional regulation, with INTS10 being essential for proper RNA processing pathways in eukaryotic cells .

What are the general characteristics of commercially available INTS10 antibodies?

INTS10 antibodies are available in several formats with varying specificities and applications:

While specific biotin-conjugated INTS10 antibodies are not directly mentioned in the search results, biotin conjugation represents an important modification strategy that enhances detection sensitivity through biotin-streptavidin interactions, similar to what's observed with other antibodies like IP10/CXCL10 .

How should researchers optimize Western blot protocols for INTS10 detection?

For optimal Western blot detection of INTS10:

• Use recommended dilutions of 1:1000-1:3000 for INTS10 primary antibodies

• Account for potential molecular weight variations (70-82 kDa observed vs. 82 kDa calculated)

• Include appropriate positive controls from validated cell lines (MCF-7 cells) or tissues (rat testis, mouse kidney)

• When using biotin-conjugated antibodies, implement streptavidin-HRP or streptavidin-fluorophore detection systems for signal enhancement

• Consider using PVDF membranes rather than nitrocellulose to improve protein retention

• Extend primary antibody incubation to overnight at 4°C to maximize binding efficiency

Published research has validated INTS10 antibodies (Proteintech 15271-1-AP) for Western blotting at concentrations of 0.4 μg/ml in human samples, as reported in Nature Communications (2014) .

What considerations are important for immunoprecipitation experiments with INTS10 antibodies?

For successful immunoprecipitation of INTS10:

• Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

• Select appropriate cell lines with confirmed INTS10 expression (e.g., HeLa cells show positive IP results)

• For biotin-conjugated antibodies, utilize streptavidin-coated beads rather than Protein A/G

• Consider crosslinking strategies to reduce heavy/light chain interference in subsequent analyses

• Perform IP under native conditions to preserve protein-protein interactions within the Integrator complex

• Include RNase inhibitors if investigating RNA-protein interactions involving INTS10

How can researchers validate the specificity of INTS10 antibodies in their experimental systems?

Comprehensive validation approaches include:

• Knockdown/knockout controls: Publications using INTS10 knockdown/knockout models have been documented and represent the gold standard for validation

• Molecular weight verification: Confirm detection within the expected 70-82 kDa range

• Peptide competition assays: Particularly relevant for antibodies raised against synthetic peptides, such as the C-terminal region-specific antibody (amino acids 523-553)

• Cross-species reactivity assessment: Verify consistent detection patterns across human, mouse, and rat samples if using for comparative studies

• Orthogonal technique confirmation: Compare results across Western blot, immunohistochemistry, and immunoprecipitation

• Recombinant protein controls: Use purified INTS10 protein as a positive control for antibody specificity

What are the optimal storage and handling conditions for INTS10 antibodies?

To maintain antibody functionality:

• Store unconjugated INTS10 antibodies at -20°C for long-term storage (stable for one year after shipment)

• For short-term use (up to one month), storage at 2-8°C is acceptable for some formulations

• For biotin-conjugated antibodies, aliquot to minimize freeze-thaw cycles

• Many INTS10 antibody formulations come in stabilizing buffers with glycerol (50%) and sodium azide (0.02%)

• Some formulations may contain BSA (0.1%) as a stabilizer in smaller sizes

• Avoid repeated freeze-thaw cycles, particularly for conjugated antibodies

What advantages does biotin conjugation offer for INTS10 antibody applications?

Biotin conjugation provides several experimental advantages:

• Signal amplification: The high-affinity biotin-streptavidin interaction (Kd ≈ 10^-15 M) enhances detection sensitivity for low-abundance targets

• Multiplexing capability: Enables simultaneous detection of multiple targets when combined with other detection systems

• Versatility across platforms: Compatible with multiple detection systems including fluorescence, chemiluminescence, and enzymatic methods

• Reduced background: Circumvents species cross-reactivity issues common with secondary antibodies

• Enhanced stability: Biotin conjugation often increases antibody shelf-life compared to enzymatic conjugations

• Flexibility in experimental design: Enables detection using various streptavidin-conjugated reporters (HRP, fluorophores, gold particles)

How should researchers troubleshoot non-specific binding with biotin-conjugated INTS10 antibodies?

When encountering non-specific binding:

• Pre-block endogenous biotin in samples using avidin/streptavidin blocking kits, particularly important for tissues with high biotin content

• Optimize antibody concentration - titrate to find the minimal effective concentration

• Include appropriate blocking reagents that contain biotin-free proteins (e.g., casein-based blockers rather than BSA)

• When performing multiplex assays, carefully select compatible detection systems to avoid cross-reactivity

• Consider using biotin-conjugated F(ab')2 fragments rather than whole IgG to reduce Fc receptor-mediated binding

• Implement stringent washing steps with detergent-containing buffers to reduce non-specific interactions

What considerations are important when using INTS10 antibodies across different species?

For cross-species applications:

• Several INTS10 antibodies show validated reactivity with human, mouse, and rat samples

• The observed cross-reactivity suggests conserved epitopes across mammalian species

• For biotin-conjugated antibodies, species considerations may be less critical since detection relies on the biotin-streptavidin interaction rather than species-specific secondary antibodies

• When designing studies across species, verify sequence homology in the antibody's target region

• Consider differences in INTS10 expression levels across tissues of different species

• Validate each new species application even when cross-reactivity is expected

How can researchers incorporate INTS10 antibodies in multi-parameter analyses?

For complex experimental designs:

• Biotin conjugation enables integration into multiplex flow cytometry panels using streptavidin-fluorophore conjugates

• When combining with other biotin-containing detection systems, implement sequential staining protocols to avoid competition

• For co-localization studies, select compatible fluorophores for streptavidin conjugates that minimize spectral overlap

• In multi-omics approaches, INTS10 antibodies can be used for immunoprecipitation prior to mass spectrometry analysis

• When studying the full Integrator complex, consider multiplexed immunoprecipitation approaches targeting different complex components

• For chromatin studies, optimize protocols for chromatin immunoprecipitation (ChIP) to investigate INTS10's role in transcriptional regulation