ICMTB Antibody

What is the ICMT antibody and what are its primary applications?

The ICMT antibody is a rabbit polyclonal antibody that specifically targets isoprenylcysteine carboxylmethyltransferase (ICMT), a 32 kDa protein involved in post-translational modification of prenylated proteins. This antibody has been validated for Western blot (WB) applications at dilutions of 1:500-1:2,000 and immunohistochemistry (IHC) at dilutions of 1:50-1:200. The antibody demonstrates reactivity against human, mouse, and rat ICMT protein, making it suitable for comparative studies across these species. The immunogen used for antibody production is a recombinant fusion protein containing a sequence corresponding to amino acids 175-284 of human ICMT (NP_036537.1) .

When designing experiments with this antibody, researchers should account for its polyclonal nature, which provides broader epitope recognition but may introduce batch-to-batch variability. For optimal results, validation experiments should be performed for each new lot received.

What is the iMab antibody and how does it function in nucleic acid research?

The iMab antibody is a specialized antibody developed to selectively recognize i-Motif (iM) structures, which are quadruplex nucleic acid conformations that form in cytosine-rich regions. Traditionally, i-Motifs were thought to form only in vitro due to their acidic pH dependence, but the development of the iMab antibody has enabled researchers to detect these structures in cells, revealing their presence at gene promoters and their cell cycle dependence .

The iMab antibody has been demonstrated to selectively bind to both intramolecular and intermolecular i-Motif structures, making it a valuable tool for investigating the biological roles of these unusual DNA conformations. Recent research has confirmed the specificity of the iMab antibody and clarified that it recognizes the unique structural features of i-Motifs rather than simply binding to cytosine-rich sequences regardless of conformation .

How should ICMT antibody be stored and handled for optimal performance?

The ICMT antibody is supplied in Phosphate Buffered Saline (pH 7.3) with 50% Glycerol and 0.02% Sodium Azide. For shipping, the antibody is maintained at 4°C. Upon receipt, it should be aliquoted to minimize freeze-thaw cycles and stored at -20°C for long-term preservation .

When working with this antibody, researchers should:

• Avoid repeated freeze-thaw cycles, which can compromise antibody performance

• Thaw aliquots completely before use and gently mix by inversion or gentle pipetting

• Briefly centrifuge tubes after thawing to collect all liquid

• Keep the antibody on ice when in use during extended experimental procedures

• Return unused portion to -20°C as soon as possible

These handling protocols will help maintain antibody specificity and sensitivity, particularly for quantitative applications like Western blotting.

How does buffer composition affect the selectivity of iMab antibody binding, and what are the optimal conditions for detecting i-Motif structures?

Recent research has revealed that buffer composition significantly influences the selectivity of iMab antibody binding to i-Motif structures. The composition of buffers used during both binding and washing steps has been shown to strongly impact antibody selectivity .

To optimize iMab antibody performance in experimental settings, researchers should:

• Carefully control DNA concentrations to avoid artifacts caused by intermolecular interactions at high concentrations

• Optimize blocking conditions to minimize non-specific binding

• Select appropriate buffer compositions for both binding and washing steps

• Consider the potential formation of intermolecular i-Motifs when working with C-rich sequences

Nuclear magnetic resonance (NMR) studies have demonstrated that several C-rich sequences previously not expected to form i-Motifs can actually form intermolecular i-Motifs that are selectively recognized by the iMab antibody . This highlights the importance of understanding the structural dynamics of target sequences when interpreting iMab binding results.

What are the challenges in targeting intracellular antigens with antibodies like ICMT, and what delivery strategies exist?

Targeting intracellular antigens like ICMT with antibodies presents significant challenges due to the cell membrane barrier. Unlike membrane-bound or secreted proteins, intracellular targets require specialized delivery strategies to enable antibody access while maintaining functionality .

Several approaches for intracellular antibody delivery have been developed:

• Protein-transduction domains or their mimics: These peptide sequences can facilitate membrane penetration

• Liposomal delivery systems: Encapsulation in liposomes can enhance cellular uptake

• Polymer vesicles: These can protect antibodies during delivery and facilitate release

• Viral envelope-based systems: Modified viral components can be used for delivery

• Single-domain antibodies: Smaller antibody formats like VHH antibodies (Nanobodies®) may have improved intracellular penetration

When designing experiments targeting intracellular ICMT, researchers should consider these delivery options and evaluate their compatibility with downstream applications. Each approach has distinct advantages and limitations regarding efficiency, specificity, and potential effects on cellular physiology.

How can researchers distinguish between true iMab antibody binding to i-Motif structures versus potential artifacts in experimental settings?

Distinguishing between genuine iMab binding to i-Motif structures and experimental artifacts requires careful experimental design and appropriate controls. Recent research has identified several key considerations:

• Buffer composition: The composition of buffers used during binding and washing steps strongly influences binding selectivity

• DNA concentration: High DNA concentrations can promote intermolecular i-Motif formation, potentially leading to misinterpretation of results

• Washing conditions: Stringent washing protocols can help differentiate between specific and non-specific binding

• Structural validation: Complementary techniques such as NMR should be employed to confirm i-Motif formation

To minimize artifacts, researchers should:

• Include appropriate negative controls (sequences unlikely to form i-Motifs)

• Use positive controls with known i-Motif-forming capability

• Employ multiple detection methods to validate findings

• Consider pH conditions, as i-Motifs are pH-dependent structures

• Validate findings with orthogonal structural biology techniques

What are the optimal Western blot protocols when using ICMT antibody?

For successful Western blot applications with ICMT antibody, researchers should follow these methodological recommendations:

• Sample preparation:

  • Use fresh tissue/cell lysates when possible

  • Include protease inhibitors in lysis buffers

  • Denature samples thoroughly at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 32 kDa ICMT protein

  • Transfer to PVDF membranes at 100V for 60-90 minutes or 30V overnight at 4°C

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour

  • Dilute ICMT antibody 1:500-1:2,000 in blocking buffer

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash membranes thoroughly (4 x 5 minutes) with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5,000-1:10,000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) reagents for detection

  • Expose to film or capture using a digital imaging system

If non-specific bands appear, consider increasing blocking time/concentration, optimizing antibody dilution, or adding 0.1% Tween-20 to antibody dilution buffer to reduce background.

How can researchers optimize protocols for detecting i-Motif structures using iMab antibody in cellular contexts?

Detecting i-Motif structures in cellular contexts using iMab antibody requires careful optimization to ensure specificity and sensitivity. Recommended methodological approaches include:

• Sample preparation:

  • Fix cells using paraformaldehyde (2-4%) to preserve nuclear architecture

  • Permeabilize with 0.1-0.5% Triton X-100 to allow antibody access

  • Consider native conditions to preserve DNA structure

• Blocking:

  • Use 2-5% BSA in PBS with 0.1% Tween-20

  • Include salmon sperm DNA or other non-specific DNA competitors to reduce background

• Antibody incubation:

  • Optimize iMab concentration through titration experiments

  • Incubate samples overnight at 4°C

  • Include stringent washing steps with optimized buffer compositions

• Controls:

  • Include cells treated with DNase as negative controls

  • Consider pH manipulation to alter i-Motif formation as control conditions

  • Include sequences known to form or not form i-Motifs

• Detection:

  • Use fluorescently labeled secondary antibodies for immunofluorescence

  • Consider super-resolution microscopy for detailed localization studies

When interpreting results, researchers should be mindful that buffer composition during binding and washing steps strongly influences the selectivity of antibody binding .

What strategies exist for validating the specificity of antibodies like ICMT and iMab in research applications?

Validating antibody specificity is crucial for reliable experimental outcomes. For ICMT and iMab antibodies, consider these validation approaches:

For ICMT antibody:

• Genetic validation:

  • Use ICMT knockout or knockdown cell lines as negative controls

  • Compare antibody reactivity in wild-type versus modified systems

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific binding should be competitively inhibited

• Multiple antibody approach:

  • Use alternative antibodies targeting different ICMT epitopes

  • Consistent results with different antibodies increase confidence

For iMab antibody:

• Structural validation:

  • Use NMR or circular dichroism to confirm i-Motif formation in target sequences

  • Compare antibody binding with structural confirmation

• Sequence specificity:

  • Test antibody binding to sequences with systematic mutations

  • Identify critical nucleotides for structure formation and antibody recognition

• pH dependence:

  • Exploit the pH-dependence of i-Motif formation

  • Compare binding at different pH values where the structure forms or dissolves

• Buffer optimization:

  • Test different buffer compositions during binding and washing steps

  • Identify conditions that maximize specific binding while minimizing artifacts

What are the detailed specifications of commercially available ICMT antibody?

The following table provides comprehensive specifications for commercially available ICMT antibody:

This antibody is supplied as a research tool and is not intended for diagnostic or therapeutic use .

How can intracellular antibody delivery techniques be applied to ICMT antibody research?

Intracellular delivery of antibodies like ICMT presents significant opportunities for studying protein function in native cellular environments. Several advanced techniques can be applied to ICMT antibody research:

• Protein transduction domains (PTDs):

  • Conjugate PTDs such as TAT, penetratin, or polyarginine to ICMT antibodies

  • These positively charged sequences interact with cell membrane components to facilitate internalization

  • Monitor potential effects on antibody binding capacity post-conjugation

• Liposomal delivery systems:

  • Encapsulate ICMT antibodies in cationic liposomes

  • Optimize lipid composition for specific cell types

  • Consider pH-sensitive formulations for endosomal escape

• Electroporation:

  • Apply electrical pulses to create temporary pores in cell membranes

  • Optimize voltage and pulse duration for different cell types

  • Balance delivery efficiency with cell viability

• Microinjection:

  • Direct injection of antibodies into cells for precise delivery

  • Useful for single-cell studies but low throughput

  • Requires specialized equipment and operator skill

• Antibody engineering approaches:

  • Consider smaller antibody formats like single-domain antibodies or nanobodies

  • These formats (approximately 15 kDa) may have enhanced cellular penetration

  • VHH antibodies lacking disulfide bonds have shown good stability and solubility

When applying these techniques, researchers should validate that:

• The antibody maintains specificity after delivery

• The delivery process itself doesn't affect cellular processes being studied

• Sufficient antibody concentration reaches the intended intracellular compartment

What applications exist for T-cell receptor mimic (TCRm) antibodies in targeting intracellular tumor antigens?

TCRm antibodies represent an innovative approach to targeting intracellular antigens for therapeutic purposes. Unlike traditional antibodies that typically target cell surface proteins, TCRm antibodies recognize peptide-MHC-I complexes displayed on cell surfaces, where the peptides are derived from intracellular proteins .

Key applications and considerations for TCRm antibodies include:

• Cancer immunotherapy:

  • TCRm antibodies can recognize tumor-associated peptide-MHC-I complexes

  • This approach enables targeting of intracellular oncoproteins previously considered "undruggable"

  • The specificity of antibody recognition provides potential advantages over small molecule approaches

• Mechanism of action:

  • TCRm antibodies can mediate effects through multiple pathways:

  • Fc-mediated immune effector functions (antibody-dependent cellular cytotoxicity)

  • Complement-dependent cytotoxicity

  • Direct signaling effects upon binding

• Target selection considerations:

  • Peptide presentation levels on different tumor types

  • MHC restriction (limiting application to patients with specific HLA types)

  • Potential for off-target effects on normal tissues expressing the target

• Advantages compared to TCR-based therapies:

  • Potentially better safety profile

  • More stable molecules with longer half-lives

  • No requirement for patient lymphocyte extraction

• Current research status:

  • Multiple TCRm antibodies have been tested in vitro and in vivo

  • Expanding understanding of mechanisms of action

  • Importance of target epitope selection and expression levels

When considering TCRm antibodies for research or therapeutic development, careful validation of target peptide presentation, specificity testing, and assessment of potential cross-reactivity are essential.