MOBP Antibody, Biotin conjugated

What is MOBP and why is it an important research target?

MOBP (Myelin Associated Oligodendrocyte Basic Protein) is a 21 kDa protein consisting of 183 amino acid residues in humans that is primarily localized in the cytoplasm. It plays a critical role in compacting or stabilizing the myelin sheath, likely by binding to negatively charged acidic phospholipids of the cytoplasmic membrane . MOBP is notably expressed in the cerebral cortex and serves as an important marker for identifying Brain Oligodendrocyte Precursor Cells and mature Brain Oligodendrocytes .

The protein is particularly significant for researchers studying myelination processes, demyelinating disorders, and oligodendrocyte function. Up to four different isoforms have been reported for this protein, with orthologs documented in mouse, rat, bovine, and chimpanzee species . This conservation across species makes MOBP an excellent target for comparative neurological research.

What are the key characteristics of MOBP Antibody, Biotin conjugated?

MOBP Antibody, Biotin conjugated (such as ABIN1389775) is a polyclonal antibody typically raised in rabbits that targets specific amino acid sequences of the MOBP protein. The specific binding region is often the amino acid sequence 7-90 . This antibody demonstrates reactivity across multiple species including human, mouse, and rat samples .

The biotin conjugation involves covalent cross-linking of biotin to the epsilon-amino groups of lysine residues in the antibody using an N-succinimidyl ester . This conjugation enables detection through avidin-based systems by exploiting the high-affinity interaction between biotin and avidin/streptavidin. The antibody is typically supplied in an aqueous buffered solution containing TBS (pH 7.4) with BSA, a preservative like ProClin, and glycerol .

How does biotinylation affect antibody functionality in research applications?

Specifically, biotinylated monoclonal antibodies are substantially weaker in causing classical complement pathway-mediated lysis of target cells compared to their non-biotinylated counterparts . This occurs because biotinylation blocks the binding of C1q to the antibody Fc-regions, preventing activation of the classical complement pathway . This characteristic has important implications for functional studies involving complement activation.

For detection-based applications (WB, ELISA, IHC), this limitation is generally not problematic and may even be advantageous when unwanted complement activation could interfere with experimental results. The high-affinity interaction between biotin and avidin/streptavidin provides a robust detection system that can enhance sensitivity in many applications.

What protocols are recommended for immunohistochemistry using MOBP Antibody, Biotin conjugated?

For immunohistochemistry applications using MOBP Antibody, Biotin conjugated, researchers should follow these methodological guidelines:

For Paraffin-embedded Sections (IHC-P):

• Sample Preparation: Fix tissue in 10% neutral buffered formalin, process, embed in paraffin, and section at 4-6 μm thickness.

• Deparaffinization: Treat slides with xylene and rehydrate through graded alcohols to water.

• Antigen Retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0).

• Endogenous Peroxidase Block: Incubate sections in 3% hydrogen peroxide for 10 minutes.

• Blocking: Apply 5% normal serum (from the same species as the secondary antibody) for 30 minutes.

• Primary Antibody: Apply MOBP Antibody, Biotin conjugated at a dilution of 1:200-400 and incubate overnight at 4°C .

• Detection: Apply streptavidin-HRP complex and visualize using DAB substrate.

• Counterstain: Apply hematoxylin, dehydrate, clear, and mount.

For Frozen Sections (IHC-F):

For frozen sections, follow a similar protocol but with a primary antibody dilution of 1:100-500 . Omit the deparaffinization and antigen retrieval steps, and fix sections briefly in acetone or 4% paraformaldehyde before beginning the staining protocol.

What are the optimization strategies for Western blotting with MOBP Antibody, Biotin conjugated?

Western blotting with MOBP Antibody, Biotin conjugated requires careful optimization to detect the 21 kDa MOBP protein effectively. Follow these methodological considerations:

• Sample Preparation: Extract proteins from tissue samples (preferably brain tissue) using a buffer containing protease inhibitors. For MOBP detection, brain tissue from cerebral cortex is recommended based on expression patterns .

• Protein Loading: Load 20-50 μg of total protein per lane. Include both positive controls (brain tissue) and negative controls.

• Gel Percentage: Use 12-15% polyacrylamide gels to effectively resolve the 21 kDa MOBP protein.

• Transfer Conditions: Transfer to PVDF membranes (preferred over nitrocellulose for small proteins) using standard transfer buffer at 100V for 1 hour or 30V overnight at 4°C.

• Blocking: Block membranes with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature.

• Primary Antibody: Dilute MOBP Antibody, Biotin conjugated at 1:300-5000 in blocking solution and incubate overnight at 4°C .

• Detection Options:

  • Direct detection with streptavidin-HRP (1:2000-1:5000)

  • Amplified detection using streptavidin-biotin-HRP complex system

• Visualization: Develop using enhanced chemiluminescence (ECL) substrate and image with a digital imaging system.

• Stripping and Reprobing: If needed, strip membranes using commercial stripping buffer and reprobe with loading control antibodies.

How does biotinylation affect complement activation when using antibodies?

Biotinylation significantly impairs the ability of antibodies to activate the classical complement pathway. Research has shown that biotinylated monoclonal antibodies are substantially weaker in causing classical complement pathway-mediated lysis of target cells compared to their non-biotinylated forms . This effect has been documented across multiple antibody types, including those targeting glycophorin A, CD59, and gangliosides .

The mechanism behind this limitation has been elucidated through binding assays using 125I-labeled C1q. These studies demonstrate that significantly less C1q binds to biotinylated antibodies compared to non-biotinylated antibodies . This occurs because the biotinylation process, which involves covalent cross-linking of biotin to the epsilon-amino groups of lysine residues, blocks the binding sites for C1q on the antibody Fc-regions .

Importantly, while biotinylation inhibits complement activation, it does not affect the antibody's ability to bind to its target antigen . Additionally, specialized functions like the ability of anti-CD59 antibodies to neutralize the complement lysis-restrictive effect of CD59 remain intact despite biotinylation .

What cross-reactivity considerations should researchers address when using MOBP Antibody, Biotin conjugated?

When working with MOBP Antibody, Biotin conjugated, researchers should consider the following cross-reactivity aspects:

• Species Cross-Reactivity: The antibody demonstrates confirmed reactivity with human, mouse, and rat MOBP . Additionally, predicted reactivity has been reported for dog, cow, sheep, pig, and horse samples, though these require validation in each laboratory setting .

• Isoform Recognition: MOBP exists in up to four different isoforms . Depending on the epitope recognized by the antibody (e.g., AA 7-90), some isoforms may be detected while others might not. Researchers should verify which isoforms are relevant to their study.

• Potential Cross-reactivity with Other Myelin Proteins: The myelin sheath contains multiple basic proteins that share some structural similarities. Validation experiments should be performed to ensure specificity, particularly when working with whole brain lysates or tissue sections.

• Endogenous Biotin Consideration: Certain tissues, particularly liver, kidney, and brain, contain endogenous biotin that can lead to false-positive signals when using biotin-streptavidin detection systems. Blocking endogenous biotin or using alternative detection methods may be necessary in these cases.

What are the storage and handling requirements for maintaining optimal activity of MOBP Antibody, Biotin conjugated?

To maintain the activity and specificity of MOBP Antibody, Biotin conjugated, researchers should adhere to the following storage and handling guidelines:

• Storage Temperature: Store the antibody at -20°C for long-term stability. The shelf life under these conditions is typically 12 months from the date of receipt .

• Aliquoting: Upon receipt, prepare small single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody performance. Each freeze-thaw cycle can reduce activity by approximately 10%.

• Buffer Composition: The antibody is typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% ProClin300, and 50% Glycerol . This formulation helps maintain stability during storage.

• Working Solution Preparation: When preparing working dilutions, use fresh buffer free of contamination. For extended use of diluted antibody (>24 hours), add a carrier protein such as BSA (0.1-1%) and store at 4°C.

• Safety Precautions: Note that the preservative ProClin is classified as poisonous and hazardous and should be handled by trained staff with appropriate safety measures .

• Shipping Conditions: The antibody can tolerate ambient temperatures during shipping for short periods, but should be stored at -20°C immediately upon receipt.

How can background signal be reduced when using MOBP Antibody, Biotin conjugated in immunohistochemistry?

Background signal is a common challenge when using biotin-conjugated antibodies in immunohistochemistry. Researchers can implement the following methodological approaches to reduce background:

• Block Endogenous Biotin: Tissues, particularly brain tissue, contain endogenous biotin that can cause high background. Use a commercial avidin/biotin blocking kit before applying the primary antibody. This typically involves sequential incubation with avidin and biotin solutions.

• Optimize Antibody Dilution: Test various dilutions of the MOBP Antibody, Biotin conjugated, starting with the recommended range (1:200-400 for IHC-P and 1:100-500 for IHC-F) . Higher dilutions may reduce background while maintaining specific signal.

• Extend Blocking Step: Increase blocking time to 1-2 hours using 5-10% normal serum from the same species as the secondary reagent, combined with 1% BSA in PBS or TBS.

• Use of Detergents: Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to blocking and antibody diluent solutions to reduce non-specific hydrophobic interactions.

• Optimize Streptavidin-HRP Concentration: Dilute streptavidin-HRP reagent more extensively (1:500-1:2000) to reduce non-specific binding while maintaining specific signal.

• Include Adequate Washing Steps: Perform 3-5 washes of 5 minutes each between each step using TBS-T or PBS-T (0.05% Tween-20).

• Use Specialized Blocking Reagents: For particularly challenging samples, commercial background reducers or addition of 0.1-1% non-fat dry milk to the antibody diluent may be beneficial.

What controls should be included when using MOBP Antibody, Biotin conjugated?

Rigorous experimental design requires appropriate controls to validate results obtained with MOBP Antibody, Biotin conjugated:

• Positive Tissue Control: Include tissue sections known to express MOBP (cerebral cortex or other myelinated regions of the central nervous system) . This confirms that the staining protocol works correctly.

• Negative Tissue Control: Include tissue sections that do not express MOBP (non-neural tissues) to verify the absence of non-specific binding.

• Absorption Control: Pre-incubate the antibody with purified MOBP protein or the immunogenic peptide (if available) before application to the tissue. This should eliminate specific staining.

• Isotype Control: Include a biotin-conjugated rabbit IgG (matching the MOBP antibody's host species and isotype) at the same concentration as the primary antibody to identify non-specific binding of immunoglobulins.

• No Primary Control: Process sections without the primary antibody but with all other reagents to identify background from the detection system.

• Endogenous Biotin Control: Include a control where only streptavidin-HRP is applied (omitting the biotinylated antibody) to assess background from endogenous biotin.

• Competing Methods Validation: When possible, confirm findings using an unconjugated MOBP antibody with a different detection system or another method like in situ hybridization.

How can MOBP Antibody, Biotin conjugated be used in multi-color immunofluorescence studies?

Multi-color immunofluorescence studies with MOBP Antibody, Biotin conjugated require careful methodological planning to achieve optimal results:

• Avidin-Fluorophore Selection: Use streptavidin conjugated to fluorophores with emission spectra distinct from other fluorophores in your multiplexing panel. Common choices include:

  • Streptavidin-Alexa Fluor 488 (green emission)

  • Streptavidin-Alexa Fluor 555/568 (red emission)

  • Streptavidin-Alexa Fluor 647 (far-red emission)

• Sequential Staining Protocol:
  a. Block endogenous biotin using an avidin/biotin blocking kit
  b. Apply non-biotinylated primary antibodies first (different host species from MOBP antibody)
  c. Apply appropriate species-specific secondary antibodies
  d. Block with excess unconjugated secondary antibody to prevent cross-reactivity
  e. Apply MOBP Antibody, Biotin conjugated
  f. Apply fluorophore-conjugated streptavidin

• Co-localization Studies: MOBP Antibody, Biotin conjugated is particularly valuable for co-localization studies with other myelin proteins or oligodendrocyte markers. Recommended combinations include:

  • MOBP (biotin) + MBP (myelin basic protein)

  • MOBP (biotin) + PLP (proteolipid protein)

  • MOBP (biotin) + OLIG2 (oligodendrocyte transcription factor)

• Signal Amplification Options: For low-abundance targets, consider using tyramide signal amplification (TSA) with the biotinylated antibody, which can increase sensitivity by 10-100 fold.

• Image Acquisition Considerations: When imaging, acquire channels sequentially rather than simultaneously to prevent bleed-through. Include single-stained controls for each fluorophore to establish proper exposure settings and compensation parameters.

How can MOBP Antibody, Biotin conjugated be utilized in studying demyelinating diseases?

MOBP Antibody, Biotin conjugated provides valuable tools for investigating demyelinating diseases through the following methodological approaches:

• Quantitative Analysis of Myelin Loss:

  • Use standardized immunohistochemistry protocols with MOBP Antibody, Biotin conjugated on tissue sections from disease models or patient samples

  • Employ image analysis software to quantify MOBP-positive area as a percentage of total white matter

  • Compare results across disease stages, treatment groups, or between patient and control samples

• Disease Progression Monitoring:

  • In longitudinal animal studies of demyelinating diseases, MOBP expression can be tracked at different timepoints

  • Correlate MOBP levels with clinical scores and other pathological markers

  • The biotin-streptavidin detection system offers enhanced sensitivity for detecting subtle changes in early disease stages

• Comparison with Other Myelin Proteins:

  • MOBP can be studied alongside other myelin proteins (MBP, PLP, MAG) to determine if demyelination affects different myelin components equally

  • This comparative approach helps distinguish between general demyelination and specific protein deficiencies

• Remyelination Assessment:

  • Following demyelinating events, MOBP immunostaining can track the return of myelin during recovery or after therapeutic intervention

  • The appearance of MOBP-positive oligodendrocytes at lesion edges may indicate active remyelination

• Methodological Protocol for Multiple Sclerosis Tissue Analysis:
  a. Obtain tissue sections from MS lesions and normal-appearing white matter
  b. Perform antigen retrieval using citrate buffer pH 6.0
  c. Block endogenous biotin and peroxidase
  d. Apply MOBP Antibody, Biotin conjugated (1:200 dilution) overnight
  e. Develop using streptavidin-HRP and DAB
  f. Counterstain with hematoxylin
  g. Analyze lesion centers, lesion borders, and normal-appearing white matter separately

This approach has proven particularly valuable for distinguishing between different types of MS lesions and for evaluating the efficacy of remyelination-promoting therapies.