MID1IP1 Antibody, Biotin conjugated

What is MID1IP1 and why is it significant in scientific research?

MID1IP1 (Midline 1 Interacting Protein 1) is a protein that has gained significant attention in cancer research, particularly for its role in liver cancer progression. This protein functions as a critical regulator of various cellular processes, including cancer cell proliferation, invasion, and metastasis. Research has demonstrated that MID1IP1 promotes liver cancer growth through colocalization with c-Myc, mediated by ribosomal proteins L5 and L11, establishing it as a potent oncogenic molecule . MID1IP1 is also known by multiple synonyms, including LAMTOR3, MAP2K1IP1, MAPKSP1, and PRO2783, reflecting its diverse functions across signaling pathways . Recent transcriptomic analyses have identified MID1IP1 as one of the most significantly upregulated genes in metastatic hepatocellular carcinoma (HCC), making it an important biomarker and potential therapeutic target in cancer research .

What are the key technical specifications of commercially available MID1IP1 Antibody, Biotin conjugated?

MID1IP1 Antibody, Biotin conjugated is available from multiple manufacturers with the following key specifications:

These technical specifications ensure researchers select the appropriate antibody for their experimental needs, particularly for detecting human MID1IP1 in immunoassay applications .

How does biotinylation affect the functionality and application of MID1IP1 antibodies?

Biotinylation of MID1IP1 antibodies provides significant advantages for detection sensitivity while maintaining target specificity. The biotin-streptavidin system is one of the strongest non-covalent biological interactions known, with a dissociation constant (Kd) in the femtomolar range. This property enables amplified signal detection in various experimental applications.

The biotin conjugation allows for:

• Versatile detection using streptavidin-coupled reporter molecules (e.g., HRP, fluorophores)

• Enhanced sensitivity through signal amplification strategies

• Compatibility with multiplexed detection systems

• Reduction of background in complex biological samples

What validated experimental applications are appropriate for MID1IP1 Antibody, Biotin conjugated?

MID1IP1 Antibody, Biotin conjugated has been validated for several experimental applications, though researchers should optimize conditions for their specific experimental systems:

• Enzyme Immunoassays (ELISA/EIA/RIA): The primary validated application for commercial MID1IP1 antibodies with biotin conjugation is immunoassay-based detection . These assays allow for quantitative measurement of MID1IP1 levels in various sample types.

• Immunofluorescence: Although requiring optimization, biotin-conjugated antibodies can be effectively used in immunofluorescence studies. Research by Jung et al. demonstrated successful immunofluorescence detection of MID1IP1 and its colocalization with c-Myc in hepatocellular carcinoma cells . Their protocol utilized:

  • Primary MID1IP1 antibody (1:200 dilution)

  • Secondary detection with Alexa Fluor 488 goat anti-rabbit IgG (1:500)

  • DAPI counterstaining for nuclei

  • Visualization via confocal microscopy

• Western Blotting: While Western blotting validation is not explicitly mentioned for the biotin-conjugated format, studies have used MID1IP1 antibodies for protein detection in cancer cell lysates to assess expression levels and correlate with other oncogenic markers .

• Immunohistochemistry: MID1IP1 antibodies have been successfully applied to human HCC tissue arrays to evaluate protein expression levels in clinical samples and correlate with pathological features .

Researchers should always verify the specific application validation for their selected antibody product and conduct preliminary optimization experiments.

What are the recommended storage and handling protocols to maintain optimal antibody activity?

Proper storage and handling of MID1IP1 Antibody, Biotin conjugated is essential to preserve its activity and specificity:

• Storage Temperature: Store at -20°C or below as recommended by manufacturers . Long-term storage at -80°C may further extend shelf-life.

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance .

• Light Protection: Biotin conjugates should be protected from light exposure during storage and handling to prevent photobleaching .

• Buffer Composition: The antibody is typically provided in a stabilizing buffer containing glycerol (often 50%), which prevents freezing at -20°C and maintains protein stability . Additional components may include:

  • PBS pH 7.4 (physiological buffer)

  • BSA (0.25%) as a carrier protein

  • Preservatives such as sodium azide (0.02%) or Proclin-300 (0.03%)

• Thawing Protocol: When ready to use, thaw aliquots completely at room temperature or 4°C before gentle mixing. Avoid vortexing to prevent antibody denaturation.

• Working Dilution Stability: Once diluted to working concentration, the antibody should ideally be used within 24 hours. For extended storage of diluted antibody, maintain at 4°C with appropriate preservatives.

Following these recommendations will help ensure consistent experimental results and maximize the usable lifetime of the antibody preparation .

How does MID1IP1 function in liver cancer progression and what techniques assess this role?

MID1IP1 plays a significant role in liver cancer progression through multiple mechanisms, as demonstrated by several key studies:

Functional Role in Hepatocellular Carcinoma (HCC):

• Promotes cancer cell proliferation and viability

• Enhances colony formation capacity

• Facilitates tumor growth and metastasis

• Regulates cancer cell stemness and invasive properties

Experimental Evidence and Assessment Techniques:
Jung et al. demonstrated that MID1IP1 is highly expressed in HCC cell lines (HepG2, Huh7, SK-Hep1, PLC/PRF5) compared to normal hepatocytes . Their research utilized multiple techniques to assess MID1IP1's oncogenic functions:

• Expression Analysis: Western blotting and RT-PCR revealed elevated MID1IP1 levels in HCC cells and tissues.

• Functional Assays:

  • MTT assay showed reduced viability in MID1IP1-depleted Huh7 and Hep3B cells

  • Colony formation assays demonstrated significantly reduced colony numbers in MID1IP1-depleted HepG2, Huh7, and Hep3B cells

  • Apoptosis assessment through PARP cleavage detection

• Mechanistic Investigation:

  • MID1IP1 depletion attenuated pro-PARP, c-Myc, and activated p21

  • MID1IP1 overexpression activated c-Myc and reduced p21 expression

  • Synergistic attenuation of c-Myc stability occurred with MID1IP1 depletion

The research by Wong et al. further validated MID1IP1's role in HCC metastasis using:

• Whole-transcriptome sequencing

• Orthotopic liver injection mouse models

• Tail-vein injection metastasis models

• Sphere-forming assays to assess cancer stemness

These studies collectively establish MID1IP1 as a critical promoter of liver cancer progression through multiple mechanisms, particularly through its interaction with the c-Myc oncogenic pathway.

What is the molecular mechanism of MID1IP1 and c-Myc colocalization in HCC, and how is it best studied?

The colocalization of MID1IP1 and c-Myc represents a critical molecular mechanism driving liver cancer progression. This interaction forms a central axis for cancer cell proliferation and survival.

Molecular Basis of MID1IP1-c-Myc Interaction:

• MID1IP1 depletion attenuates c-Myc expression and stability in HCC cells

• MID1IP1 overexpression activates c-Myc and reduces p21 tumor suppressor expression

• Ribosomal proteins L5 and L11 mediate this regulatory relationship

• MID1IP1 depletion upregulates L5/L11 expression, while loss of L5/L11 rescues c-Myc levels in MID1IP1-depleted cells

Optimal Methods for Studying This Colocalization:

• Confocal Immunofluorescence Microscopy:
  Jung et al. successfully visualized MID1IP1 and c-Myc colocalization using:

  • Fixed cells incubated with specific MID1IP1 antibody (1:200; Invitrogen) and c-Myc antibody (1:500; Santa Cruz)

  • Secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1:500) and Alexa Fluor 546 goat anti-mouse IgG (1:500)

  • DAPI nuclear counterstaining

  • FLUOVIEW FV10i confocal microscope and Delta Vision imaging system

• Proximity Ligation Assay (PLA):
  This technique can detect protein-protein interactions with high sensitivity by generating fluorescent signals only when proteins are within 40nm proximity.

• Co-immunoprecipitation (Co-IP):
  For biochemical verification of physical interaction between MID1IP1 and c-Myc.

• Tissue Microarray Analysis:
  Jung et al. demonstrated that MID1IP1 and c-Myc colocalization can be observed in human HCC tissues, providing clinical relevance to the interaction .

• Dual Knockdown/Overexpression Studies:
  Manipulating both MID1IP1 and c-Myc expression levels helps establish functional relationships between these proteins.

This colocalization appears to be a key mechanism of oncogenic activity, as tissue array analysis showed that MID1IP1 overexpression colocalizes with c-Myc in human HCC tissues, validating the clinical relevance of this interaction .

How does MID1IP1 expression correlate with clinical outcomes in cancer patients?

MID1IP1 expression shows significant correlation with clinical outcomes and pathological features in cancer patients, particularly in hepatocellular carcinoma (HCC). Wong et al. conducted comprehensive analyses using multiple clinical cohorts to establish these correlations :

Upregulation Pattern in Clinical Samples:

• MID1IP1 was significantly overexpressed in HCC compared to non-tumor liver tissues (NTLs)

• 47% of HCC cases in the HKU-QMH cohort (n=89) showed ≥2-fold MID1IP1 overexpression

• 50% of paired HCC cases in the TCGA cohort (n=50) demonstrated ≥2-fold upregulation

• Protein-level overexpression was confirmed by Western blotting and immunohistochemistry

Correlation with Aggressive Tumor Phenotypes:
MID1IP1 upregulation (≥2-fold) was significantly associated with multiple aggressive features:

• Presence of venous invasion

• Tumor microsatellite formation

• Direct liver invasion

• Absence of tumor encapsulation

• More advanced tumor stage

Survival Impact:

Molecular Mechanism Underlying Poor Prognosis:
Further investigation revealed that MID1IP1 promotes cancer metastasis through:

• Enhancing migratory and invasive abilities

• Increasing sphere-forming ability (cancer stemness)

• Upregulating FRA1 transcriptional activity

• Promoting MMP9-mediated extracellular matrix degradation

These clinical correlations provide strong evidence that MID1IP1 serves as both a prognostic biomarker and a mechanistic driver of aggressive disease, making it a valuable target for both diagnostic assessment and therapeutic intervention in HCC.

What are common challenges when using MID1IP1 Antibody, Biotin conjugated, and how can they be addressed?

Researchers may encounter several challenges when working with MID1IP1 Antibody, Biotin conjugated. Here are common issues and strategies to address them:

1. Weak or Absent Signal:

• Potential Causes: Insufficient antibody concentration, degraded antibody, low target expression, inefficient biotinylation

• Solutions:

  • Titrate antibody concentration (optimal dilutions should be determined experimentally)

  • Use fresh aliquots and avoid repeated freeze-thaw cycles

  • Include positive control samples with known MID1IP1 expression

  • Employ signal amplification strategies like tyramide signal amplification

2. High Background or Non-specific Binding:

• Potential Causes: Excessive antibody concentration, endogenous biotin, insufficient blocking

• Solutions:

  • Optimize antibody dilution

  • Block endogenous biotin using commercial biotin blocking kits

  • Increase blocking agent concentration (3% BSA was used successfully in Jung et al.)

  • Include additional washing steps

3. Cross-reactivity Issues:

• Potential Causes: Antibody recognizing related proteins, particularly other MAPK pathway members

• Solutions:

  • Validate with specific knockdown/knockout controls

  • Perform peptide competition assays

  • Compare multiple antibodies targeting different epitopes

4. Inconsistent Results Between Detection Methods:

• Potential Causes: Different epitope accessibility in various applications

• Solutions:

  • Optimize protocols specifically for each application

  • Consider using different antibody clones for different applications

  • Validate with appropriate controls for each method

5. Biotinylation-specific Issues:

• Potential Causes: Over-biotinylation masking epitope, streptavidin binding issues

• Solutions:

  • Use optimized commercial conjugates with controlled biotin:protein ratio

  • Test different streptavidin conjugates

  • Consider using LYNX Rapid Plus Biotin Type 1 conjugation kit for custom conjugation with controlled chemistry

When troubleshooting, maintaining detailed records of experimental conditions and systematically testing variables is essential for identifying optimal conditions for MID1IP1 detection.

What controls should be included when working with MID1IP1 Antibody, Biotin conjugated?

Proper experimental controls are essential when using MID1IP1 Antibody, Biotin conjugated to ensure reliable and interpretable results:

Essential Positive Controls:

• Known MID1IP1-expressing Samples:

  • Cell lines with confirmed high MID1IP1 expression (e.g., HepG2, Huh7, SK-Hep1)

  • Recombinant MID1IP1 protein (commercial or lab-generated)

• Parallel Detection Methods:

  • RNA expression verification (RT-qPCR)

  • Detection with alternative antibody clones

  • Protein tagging approach (e.g., FLAG/HA-tagged MID1IP1)

Critical Negative Controls:

• MID1IP1 Knockdown/Knockout Samples:

  • siRNA or shRNA-mediated MID1IP1 depletion models

  • CRISPR/Cas9-generated MID1IP1 knockout cells

  • Normal cells with naturally low MID1IP1 expression (e.g., AML-12 cells)

• Reagent Controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at matching concentration)

  • Secondary detection reagent alone

Biotin-Specific Controls:

• Endogenous Biotin Control:

  • Streptavidin-conjugate alone without primary antibody

  • Biotin blocking kit treatment comparison

• Biotinylation Efficiency Control:

  • Non-biotinylated primary antibody comparison

  • Commercial biotin-conjugated control antibody

Application-Specific Controls:

• For Western Blotting:

  • Molecular weight marker verification (MID1IP1 expected at approximately 22 kDa)

  • Loading control (e.g., β-actin, as used by Jung et al.)

• For Immunofluorescence:

  • Single-stain controls for multi-label experiments

  • Additional organelle markers for colocalization studies

• For ELISA/Immunoassays:

  • Standard curve using recombinant protein

  • Spike-in recovery tests

Including these controls provides crucial validation of antibody specificity and helps differentiate true biological findings from technical artifacts, particularly important when studying a protein involved in critical signaling pathways like MID1IP1.

What emerging techniques can be used to study MID1IP1's role in cancer signaling networks?

Advanced molecular and cellular techniques are expanding our understanding of MID1IP1's role in cancer signaling networks:

1. Multi-omics Integration Approaches:

• Proteogenomic Analysis: Combining transcriptomic data with proteomic profiling to identify post-transcriptional regulation of MID1IP1, as exemplified by Wong et al.'s integration of whole-transcriptome sequencing with protein expression analysis .

• Phosphoproteomics: Identifying MID1IP1 phosphorylation states and their impact on signaling cascades, particularly relevant given MID1IP1's role in MAPK pathways.

2. Advanced Imaging Technologies:

• Super-resolution Microscopy: Techniques like STORM or PALM can visualize MID1IP1-c-Myc interactions at nanometer resolution, beyond the diffraction limit of conventional microscopy.

• Live-cell Imaging with FRET/BRET: Monitors real-time protein-protein interactions between MID1IP1 and its binding partners in living cells.

• Spatial Transcriptomics: Combines histological analysis with gene expression profiling to map MID1IP1 expression patterns within tumor microenvironments.

3. Functional Genomics Approaches:

• CRISPR-Cas9 Screens: Genome-wide or pathway-focused screens to identify synthetic lethal interactions with MID1IP1, potentially revealing new therapeutic vulnerabilities.

• CRISPRi/CRISPRa: For precise modulation of MID1IP1 expression levels without complete knockout.

4. Pathway Analysis Technologies:

• Reverse Phase Protein Arrays (RPPA): High-throughput analysis of multiple signaling proteins simultaneously to place MID1IP1 in broader pathway contexts.

• Single-cell Phospho-Flow Cytometry: Examining MID1IP1-dependent signaling events at single-cell resolution to capture cellular heterogeneity.

5. Translational Research Approaches:

• Patient-Derived Organoids (PDOs): Testing MID1IP1 inhibition in 3D tumor models that better recapitulate in vivo conditions.

• Circulating Tumor DNA (ctDNA) Analysis: Monitoring MID1IP1 alterations in liquid biopsies as potential biomarkers.

These emerging techniques offer powerful approaches to dissect MID1IP1's complex role in cancer signaling networks and may reveal new therapeutic opportunities targeting this protein.

How is MID1IP1 regulated at the transcriptional level, and what techniques can assess this regulation?

Recent research has illuminated the transcriptional regulation of MID1IP1, particularly in the context of cancer. Wong et al. identified SP1 as a key transcriptional regulator of MID1IP1 expression :

SP1 as a Master Regulator:

• ENCODE integrative analysis predicted potential transcription factors binding to the MID1IP1 promoter

• SP1 emerged among the top 5 TFs with highest probability of binding the MID1IP1 promoter

• SP1 expression positively correlates with MID1IP1 expression in both HKU-QMH and TCGA cohorts

• This correlation was specifically observed in tumor tissues but not in normal liver tissues, suggesting cancer-specific regulation

Experimental Validation of SP1 Regulation:

• Stable SP1 knockdown (shSP1) in MHCC97L and PLC/PRF/5 cells significantly suppressed both mRNA and protein expression of MID1IP1

• Chromatin immunoprecipitation (ChIP) assay confirmed direct SP1 binding to the MID1IP1 promoter

• Significant enrichment of MID1IP1 promoter regions (−164 to −22nt and −904 to −554nt from the transcription start site) was detected in anti-SP1 immunoprecipitation

Techniques to Assess Transcriptional Regulation:

• Promoter Analysis Tools:

  • Bioinformatic prediction of transcription factor binding sites

  • Luciferase reporter assays with wild-type and mutated promoter constructs

• Chromatin-Based Methods:

  • ChIP-seq to map genome-wide TF binding patterns

  • ATAC-seq to assess chromatin accessibility at the MID1IP1 locus

  • CUT&RUN for higher resolution TF binding analysis

• Direct TF Manipulation:

  • siRNA/shRNA knockdown of candidate TFs

  • CRISPR-based transcription factor perturbation

  • Overexpression of transcription factors with validation of MID1IP1 response

• Correlation Analysis:

  • Analysis of MID1IP1 expression with TF expression across patient cohorts

  • Single-cell RNA-seq to capture heterogeneous regulation patterns

This understanding of MID1IP1's transcriptional regulation by SP1 opens potential therapeutic avenues targeting this regulatory axis in cancer, particularly hepatocellular carcinoma.

What are the downstream effectors of MID1IP1 in promoting cancer metastasis?

Wong et al. conducted an extensive investigation revealing the downstream molecular mechanisms through which MID1IP1 promotes cancer metastasis, particularly in hepatocellular carcinoma :