KIAA1191 Antibody

What is KIAA1191 protein and what are its basic characteristics?

KIAA1191 (also known as FLJ20480 or p60MONOX) is a protein with a molecular weight of approximately 33 kDa, consisting of 305 amino acids . The protein is encoded by the KIAA1191 gene (NCBI Gene ID: 57179) and has UniProt accession number Q96A73 . While its complete functional characterization is still ongoing, research suggests that KIAA1191 is involved in various cellular processes including cell growth, differentiation, and apoptosis . The protein has potential implications in cancer research and other disease mechanisms, making it a target of interest for molecular biology investigations .

What applications are KIAA1191 antibodies validated for?

KIAA1191 antibodies have been validated for multiple research applications, including:

• Western Blot (WB): Validated for detection at dilutions of 1:500-1:1000

• Immunohistochemistry (IHC): Effective at dilutions of 1:50-1:500

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Recommended at 1:200-1:800

• Immunoprecipitation (IP): Validated with certain antibodies

• ELISA: Confirmed functionality across multiple antibodies

Different antibodies may have varying performance in these applications, so researchers should refer to specific product validation data for optimal results in their experimental systems .

What species reactivity do commercially available KIAA1191 antibodies exhibit?

Current KIAA1191 antibodies demonstrate reactivity with several species:

When selecting an antibody, researchers should consider the target species in their experimental design and verify cross-reactivity in their specific application .

What are the optimal storage conditions for KIAA1191 antibodies?

Most KIAA1191 antibodies should be stored at -20°C for maximum stability and shelf life . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency . According to manufacturer recommendations, antibodies stored under these conditions are generally stable for one year after shipment . For smaller-sized antibody preparations (20 μl), some preparations may contain 0.1% BSA as a stabilizer .

What are the recommended dilutions for different experimental applications?

These ranges serve as starting points, and researchers should perform titration experiments to determine optimal concentrations for their specific samples and detection systems .

How can I validate the specificity of my KIAA1191 antibody?

Validating antibody specificity is crucial for reliable experimental results. Recommended approaches include:

• Positive and negative control tissues/cells: Use samples with known KIAA1191 expression (e.g., mouse ovary and uterus tissues have shown positive Western blot results)

• Molecular weight verification: Confirm detection at the expected molecular weight (~33 kDa)

• Knockdown/knockout validation: Use KIAA1191 siRNA (e.g., sc-91721) or CRISPR/Cas9 knockout systems (e.g., sc-412771) to create negative control samples

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of KIAA1191 to confirm consistent detection patterns

• Blocking peptide competition: If available, use the immunizing peptide to confirm signal specificity

Documenting these validation steps strengthens the reliability of experimental findings involving KIAA1191 .

What is the recommended protocol for KIAA1191 detection by Western blot?

For optimal Western blot detection of KIAA1191:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 33 kDa KIAA1191 protein

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute primary KIAA1191 antibody 1:500-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

  • Wash 3-5 times with TBST

• Detection:

  • Develop using ECL substrate

  • Expect to observe a specific band at approximately 33 kDa

Positive controls such as mouse ovary or uterus tissue lysates can be included to confirm antibody functionality .

What are the considerations for KIAA1191 detection by immunofluorescence?

For successful immunofluorescence detection of KIAA1191:

• Cell preparation:

  • Culture cells on coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Blocking and antibody incubation:

  • Block with 1-5% BSA in PBS for 30-60 minutes

  • Dilute KIAA1191 antibody 1:200-1:800 in blocking solution

  • Incubate overnight at 4°C in a humid chamber

  • Wash 3 times with PBS

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

  • Wash 3 times with PBS

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium

• Imaging considerations:

  • Use HeLa cells as a positive control, as they have been validated to show positive KIAA1191 staining

  • Include a negative control by omitting primary antibody

  • Consider co-staining with organelle markers to determine subcellular localization

Optimization of antibody dilution and fixation/permeabilization conditions may be required for different cell types .

What protocol is recommended for immunohistochemical detection of KIAA1191?

For optimal immunohistochemical detection of KIAA1191 in tissue sections:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness)

  • Deparaffinize and rehydrate through xylene and graded alcohols

• Antigen retrieval:

  • Perform antigen retrieval with TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 as an alternative

  • Heat in a pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 10-20 minutes

  • Cool to room temperature

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with serum or protein blocking solution

  • Dilute KIAA1191 antibody 1:50-1:500 in antibody diluent

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Wash with PBS or TBST

• Detection:

  • Apply appropriate secondary antibody and detection system (e.g., HRP-polymer)

  • Develop with DAB or other substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount

Human stomach tissue has been validated as a positive control for KIAA1191 immunohistochemistry .

What gene silencing tools are available for studying KIAA1191 function?

Several molecular tools are available for KIAA1191 gene silencing experiments:

When designing gene silencing experiments, researchers should consider the desired duration of silencing (transient vs. stable), cell type characteristics (transfection efficiency), and the level of knockdown required for observable phenotypes. Including appropriate controls such as non-targeting siRNA/shRNA is essential for experimental validity .

What CRISPR activation systems are available for KIAA1191 upregulation studies?

For researchers interested in upregulating KIAA1191 expression, several CRISPR activation systems are available:

These systems utilize modified CRISPR/Cas9 technology to activate endogenous KIAA1191 gene expression, which offers advantages over traditional overexpression methods as it maintains natural promoter regulation and isoform expression patterns. This approach is particularly valuable for studying dose-dependent effects of KIAA1191 in cellular processes .

What is known about KIAA1191's role in the necroptotic pathway of multiple myeloma?

Research has begun to uncover KIAA1191's involvement in the necroptotic pathway of multiple myeloma . Necroptosis is a regulated form of necrotic cell death that involves receptor-interacting protein kinases (RIPK1/RIPK3) and mixed lineage kinase domain-like protein (MLKL). In multiple myeloma, a malignancy of plasma cells, dysregulation of cell death pathways is a critical factor in disease progression and treatment resistance.

While the complete mechanistic details remain under investigation, researchers have identified that:

• KIAA1191 may interact with components of the necroptosis machinery

• Modulation of KIAA1191 expression affects sensitivity to necroptotic stimuli in multiple myeloma cells

• KIAA1191 could represent a potential therapeutic target in multiple myeloma treatment strategies

This emerging research area presents opportunities for investigating novel therapeutic approaches targeting non-apoptotic cell death mechanisms in multiple myeloma . Studies utilizing KIAA1191 gene silencing tools or antibodies for protein detection have contributed to these findings.

How can I troubleshoot weak or nonspecific KIAA1191 signal in my experiments?

When encountering challenges with KIAA1191 detection, consider these troubleshooting approaches:

• For weak signal in Western blot:

  • Increase protein loading (50-100 μg)

  • Reduce antibody dilution (try 1:250-1:500)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced detection systems (high-sensitivity ECL substrates)

  • Verify protein expression in your sample type (mouse ovary/uterus tissues serve as positive controls)

• For nonspecific bands:

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Increase washing stringency and duration

  • Titrate primary antibody concentration

  • Use freshly prepared buffers

  • Consider using a different KIAA1191 antibody targeting another epitope

• For immunohistochemistry/immunofluorescence background:

  • Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Extend blocking time

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding

  • Use more dilute antibody solution with longer incubation

  • Include appropriate controls (tissue known to be negative for KIAA1191)

• For all applications:

  • Verify antibody quality (avoid repeated freeze-thaw cycles)

  • Check expression levels in your experimental system

  • Consider antibody validation using knockdown/knockout approaches

Systematic optimization of these parameters should improve detection specificity and sensitivity .

What are the potential roles of KIAA1191 in cancer and disease mechanisms?

KIAA1191 has been implicated in several biological functions related to disease mechanisms:

• Cancer biology: Preliminary evidence suggests KIAA1191 may play roles in cell growth, differentiation, and apoptosis, key processes in cancer development and progression . Its involvement in the necroptotic pathway of multiple myeloma indicates potential roles in cancer cell death responses .

• Cell death regulation: Beyond its role in necroptosis in multiple myeloma, KIAA1191 may be involved in regulating other cell death pathways that impact disease outcomes and treatment responses.

• Cellular stress responses: KIAA1191's function may be linked to how cells respond to various stressors, potentially influencing disease states and therapeutic interventions.

Research into these areas is ongoing, and KIAA1191 represents a promising target for understanding disease mechanisms and developing potential therapeutic strategies . Future investigations may reveal more specific functions and pathways influenced by this protein.

What experimental approaches are recommended for studying KIAA1191 protein interactions?

To investigate KIAA1191 protein interactions, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use KIAA1191 antibodies validated for immunoprecipitation (e.g., sc-398723)

  • Perform reciprocal Co-IPs with antibodies against suspected interacting partners

  • Include appropriate controls (IgG, lysate from KIAA1191 knockdown cells)

• Proximity labeling approaches:

  • Generate BioID or TurboID fusion constructs with KIAA1191

  • Identify proximal proteins through biotinylation followed by streptavidin pulldown

  • Analyze interacting proteins by mass spectrometry

• Yeast two-hybrid screening:

  • Use KIAA1191 as bait to screen for interacting proteins

  • Validate interactions using alternative methods

• Fluorescence resonance energy transfer (FRET):

  • Generate fluorescent protein fusions with KIAA1191 and potential partners

  • Assess interaction through energy transfer between fluorophores

• Protein array screening:

  • Probe protein arrays with recombinant KIAA1191

  • Validate hits using Co-IP or other interaction assays

These approaches provide complementary data on KIAA1191 protein interactions, helping to elucidate its functional role in cellular processes and disease mechanisms.

How can KIAA1191 expression and localization be studied during cellular stress responses?

To investigate KIAA1191's expression and localization during cellular stress responses:

• Stress induction models:

  • Oxidative stress: H₂O₂, paraquat, or menadione treatment

  • ER stress: tunicamycin, thapsigargin, or DTT

  • Hypoxia: chemical (CoCl₂) or physical (hypoxia chamber)

  • Nutrient deprivation: serum starvation or glucose restriction

  • DNA damage: UV irradiation, cisplatin, or etoposide

• Expression analysis:

  • qRT-PCR for mRNA expression changes

  • Western blot with KIAA1191 antibodies (1:500-1:1000 dilution) for protein level changes

  • Time-course experiments to monitor expression dynamics

• Subcellular localization:

  • Immunofluorescence with KIAA1191 antibodies (1:200-1:800 dilution)

  • Co-staining with organelle markers

  • Live-cell imaging using fluorescent protein-tagged KIAA1191

  • Subcellular fractionation followed by Western blot analysis

• Post-translational modifications:

  • Phosphorylation-specific enrichment followed by Western blot

  • Mass spectrometry analysis of immunoprecipitated KIAA1191

• Functional significance:

  • KIAA1191 knockdown or knockout using available tools

  • Rescue experiments with wild-type or mutant KIAA1191

  • Assessment of cell viability, morphology, and stress response pathways