BSG Antibody

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

BSG (Basigin), also known as CD147 or EMMPRIN, is a transmembrane glycoprotein member of the immunoglobulin superfamily. It plays fundamental roles in intercellular recognition involved in immunologic phenomena, differentiation, and development. BSG is expressed by multiple cell types including epithelial cells, endothelial cells, and leukocytes. Its significance as a research target stems from its involvement in tumor progression, invasion, metastasis, inflammation, neurodegenerative diseases, and viral infections . Additionally, BSG serves as a determinant for the Ok blood group system and functions as an essential receptor on red blood cells for malaria parasites, making it relevant for infectious disease research .

What are the common applications for BSG antibodies in research?

BSG antibodies are primarily utilized in the following research applications:

• Western blot (WB): For detecting and quantifying BSG protein expression in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing BSG localization in tissue sections

• ELISA: For quantitative detection of BSG in solution

• Immunoprecipitation: For isolating BSG protein complexes

• Flow cytometry: For analyzing BSG expression in cell populations

These applications enable researchers to investigate BSG's expression patterns, protein interactions, and functional roles in various biological contexts .

What is the typical molecular weight range for BSG protein detection?

The observed molecular weight of BSG in experimental conditions typically ranges from 35-60 kDa, while the calculated molecular weight based on amino acid sequence is approximately 42 kDa . This variation is primarily due to post-translational modifications, particularly glycosylation states. When performing Western blot analysis, researchers should expect to observe BSG bands within this range, with possible variation depending on cell/tissue type and glycosylation status .

How should BSG antibodies be stored and handled for optimal performance?

For optimal performance and longevity of BSG antibodies, follow these storage and handling recommendations:

• Store lyophilized antibodies at -20°C for up to one year from the date of receipt

• After reconstitution, store at 4°C for one month or aliquot and store at -20°C for up to six months

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and performance

• Reconstitute lyophilized antibodies according to manufacturer specifications (e.g., adding 0.2ml of distilled water to yield 500μg/ml concentration)

• Store working dilutions at 4°C for short-term use (1-2 weeks)

What factors should be considered when selecting between polyclonal and monoclonal BSG antibodies?

The choice between polyclonal and monoclonal BSG antibodies depends on your specific research requirements:

Polyclonal BSG antibodies:

• Recognize multiple epitopes on the BSG protein

• Generally provide stronger signals due to binding of multiple antibodies per target molecule

• Better for applications like IHC on fixed tissues where some epitopes may be masked

• Useful for detecting proteins with low expression levels

• May show higher batch-to-batch variation

Monoclonal BSG antibodies:

• Recognize a single specific epitope

• Provide consistent results with minimal batch-to-batch variation

• Ideal for applications requiring high specificity

• Better for distinguishing between closely related proteins or isoforms

• Often preferred for therapeutic applications and reproducible assays

Consider your application, required specificity, detection sensitivity needs, and experiment reproducibility requirements when making this selection.

How can researchers validate the specificity of their BSG antibodies?

Validating antibody specificity is crucial for reliable research results. For BSG antibodies, consider these validation approaches:

• Knockout (KO) validation: Use BSG knockout cell lines or tissues as negative controls to confirm specificity

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding

• Multiple antibody concordance: Use multiple antibodies targeting different BSG epitopes and compare results

• Western blot analysis: Confirm detection of bands at the expected molecular weight (35-60 kDa)

• Recombinant protein controls: Include purified BSG protein as a positive control

• Cross-reactivity assessment: Test on samples from multiple species if cross-reactivity is claimed

KO-validated antibodies, such as CAB18032, provide additional confidence in specificity as they have already undergone rigorous validation against knockout samples .

What tissue types are most suitable for studying BSG expression patterns?

Based on research findings, these tissue types have proven valuable for studying BSG expression patterns:

• Cancer tissues: BSG is frequently overexpressed in various cancer types, making them excellent for studying its role in tumor progression

  • Liver cancer

  • Endometrial carcinoma

  • Ovarian serous adenocarcinoma

  • Lung adenocarcinoma

• Normal tissues with known BSG expression:

  • Placenta (shows consistent BSG expression)

  • Red blood cells (crucial for malaria research)

  • Brain tissues (for neurodegenerative disease studies)

• Cell lines with verified BSG expression:

  • HeLa cells

  • HepG2 cells

  • 293T cells

When selecting tissues, consider using paired normal and pathological samples from the same patient to enable direct comparison of expression changes.

What are the optimal conditions for Western blot detection of BSG?

For optimal Western blot detection of BSG, follow these technical recommendations:

Note that BSG typically appears as bands between 35-60 kDa, with potential variation due to different glycosylation states and processing forms .

How can researchers optimize IHC protocols for BSG detection in different tissue types?

Optimizing IHC for BSG detection requires attention to several key parameters:

• Antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has proven effective for BSG detection in various tissue types

• Blocking conditions: Use 10% goat serum to minimize background staining

• Primary antibody concentration: 2 μg/ml is typically effective; incubate overnight at 4°C

• Secondary antibody: Peroxidase-conjugated anti-rabbit IgG, incubated for 30 minutes at 37°C

• Visualization: DAB (3,3'-diaminobenzidine) chromogen provides good contrast for BSG detection

• Controls: Include both positive controls (tissues known to express BSG) and negative controls (primary antibody omitted)

• Tissue-specific considerations:

  • Cancer tissues may require shorter antigen retrieval times due to potentially higher BSG expression

  • Normal tissues might benefit from signal amplification systems for detection of lower expression levels

What approaches can be used to study BSG protein-protein interactions?

BSG interacts with various proteins to mediate its diverse biological functions. To study these interactions:

• Co-immunoprecipitation (Co-IP): Pull down BSG using anti-BSG antibodies and identify interacting partners by Western blot or mass spectrometry

• Proximity ligation assay (PLA): Visualize and quantify protein interactions in situ with subcellular resolution

• ELISA-based interaction assays: Measure direct binding between BSG and potential partners

• Inhibition studies: Use antibodies like MEM-M6/4 and MEM-M6/8 that can disrupt specific interactions (e.g., RH5-BSG interaction in malaria research)

• Yeast two-hybrid screens: Identify novel BSG-interacting proteins

• Surface plasmon resonance (SPR): Determine binding kinetics and affinity constants for BSG interactions

These approaches can provide complementary information about the composition, dynamics, and functional significance of BSG protein complexes.

How can researchers address non-specific binding when using BSG antibodies?

Non-specific binding can compromise experimental results. To minimize this issue:

• Optimize blocking conditions: Try different blocking agents (BSA, casein, commercial blocking buffers) if 5% milk is insufficient

• Increase washing stringency: Add additional wash steps or increase detergent concentration (0.1-0.3% Tween-20)

• Titrate antibody concentration: Test a range of dilutions to identify optimal signal-to-noise ratio

• Pre-adsorb the antibody: Incubate with proteins from a species different from your target to remove cross-reactive antibodies

• Use more specific detection methods: Consider using highly cross-adsorbed secondary antibodies

• Add protein competitors: Include non-relevant proteins (e.g., BSA) in antibody dilution buffers

• Verify antibody specificity: Some anti-BSG antibodies have been specifically tested for cross-reactivity with other proteins and confirmed to be highly specific

What approaches can address variable BSG detection across different experimental samples?

Variability in BSG detection can stem from several factors. Address these issues using:

• Normalize protein loading: Use housekeeping proteins (β-actin, GAPDH) as loading controls

• Account for glycosylation heterogeneity: BSG exists in different glycosylation states (35-60 kDa); enzymatic deglycosylation can reduce variability

• Standardize sample preparation: Use consistent lysis buffers and protein extraction protocols

• Include positive controls: Run known BSG-expressing samples (e.g., HeLa, HepG2 cell lysates) alongside experimental samples

• Optimize detection for different cell/tissue types: Different tissues may require adjusted antibody concentrations

• Consider isoform-specific detection: BSG has multiple isoforms; some antibodies may detect specific isoforms better than others

• Use quantitative methods: Consider quantitative Western blot or ELISA for more precise comparisons

How can researchers differentiate between various BSG isoforms?

BSG exists in multiple isoforms with distinct functional properties. To differentiate between them:

• Select antibodies recognizing specific regions: Choose antibodies targeting regions that differ between isoforms

• Use high-resolution gel systems: Employ gradient gels (5-20%) for better separation of closely migrating isoforms

• Combine with molecular techniques: Use RT-PCR with isoform-specific primers to correlate protein detection with mRNA expression

• Employ 2D electrophoresis: Separate BSG isoforms based on both molecular weight and isoelectric point

• Mass spectrometry analysis: Identify specific peptides unique to each isoform

• Use recombinant isoform standards: Include purified BSG isoforms as reference controls

• Isoform-specific knockdown: Use siRNA targeting specific isoforms to confirm antibody specificity

How can BSG antibodies be utilized in cancer research applications?

BSG is implicated in cancer progression through multiple mechanisms. Researchers can leverage BSG antibodies in cancer studies through:

• Prognostic marker evaluation: Assess correlation between BSG expression levels and patient outcomes across different cancer types

• Therapeutic target validation: Use antibodies to block BSG function in vitro and in vivo to evaluate anti-tumor effects

• Mechanism studies: Investigate how BSG promotes tumor invasion by inducing matrix metalloproteinase expression

• Cancer stem cell research: Examine BSG's role in maintaining cancer stem cell properties

• Drug resistance mechanisms: Study BSG's involvement in chemoresistance

• Metastasis research: Track BSG expression during epithelial-mesenchymal transition and metastatic spread

• Tumor microenvironment analysis: Investigate BSG's role in tumor-stroma interactions

IHC studies using anti-BSG antibodies have successfully detected BSG in various cancer tissues including liver cancer, endometrial carcinoma, ovarian serous adenocarcinoma, and lung adenocarcinoma .

What are the considerations for using BSG antibodies in infectious disease research, particularly malaria?

BSG serves as an essential receptor for Plasmodium falciparum RH5 protein during malaria parasite invasion of erythrocytes. When using BSG antibodies in malaria research:

• Invasion inhibition assays: Evaluate antibodies for their ability to block parasite invasion by disrupting RH5-BSG interaction

• Binding interaction studies: Use antibodies like MEM-M6/4 and MEM-M6/8 that have demonstrated inhibitory effects on RH5-BSG interaction

• Epitope mapping: Identify which BSG epitopes are critical for parasite binding

• Cross-reactivity considerations: Ensure antibodies recognize BSG across relevant species if conducting animal model studies

• Combination approaches: Test BSG antibodies alongside other invasion-blocking antibodies for synergistic effects

• Ex vivo studies: Apply BSG antibodies in parasite culture systems to evaluate invasion efficiency

• Structural studies: Use antibodies to probe BSG conformational changes during parasite engagement

How can researchers develop and characterize novel anti-BSG antibodies for specialized applications?

For researchers developing new anti-BSG antibodies for specific applications:

• Immunogen design: Select target regions based on:

  • Functional domains of interest

  • Regions with high antigenicity and surface exposure

  • Conserved sequences for cross-species reactivity

  • Unique sequences for isoform specificity

• Production approaches:

  • Hybridoma technology for monoclonal antibodies

  • Recombinant antibody engineering for chimeric, humanized, or fully human antibodies

  • Phage display for selecting high-affinity binders

• Validation strategy:

  • Confirm binding to recombinant and native BSG

  • Assess specificity using knockout controls

  • Characterize using multiple techniques (ELISA, WB, IHC, flow cytometry)

  • Determine functional effects on known BSG interactions

• Functional characterization:

  • Test for effects on BSG-mediated cellular processes

  • Map epitopes using peptide arrays or mutagenesis

  • Determine binding kinetics using surface plasmon resonance

How are BSG antibodies being employed in neurodegenerative disease research?

BSG has emerging roles in neurodegenerative conditions, and researchers are utilizing antibodies to:

• Map expression patterns: Examine BSG distribution in normal vs. diseased brain tissues

• Study blood-brain barrier function: Investigate BSG's role in transporter regulation at the BBB

• Analyze neuroinflammatory processes: Examine BSG's contribution to microglial activation and neuroinflammation

• Explore therapeutic possibilities: Test whether blocking BSG function modulates disease progression

• Investigate protein aggregation: Study potential interactions between BSG and disease-associated proteins (e.g., amyloid-β, tau)

• Examine metabolic regulation: Assess BSG's role in neural energy metabolism through MCT transporter regulation

• Develop biomarker applications: Evaluate BSG as a potential biomarker for disease progression

What are the methodological approaches for studying BSG in extracellular vesicles and intercellular communication?

BSG is enriched in extracellular vesicles (EVs) and plays roles in intercellular communication. To study these functions:

• EV isolation and characterization:

  • Isolate EVs using ultracentrifugation, size exclusion chromatography, or commercial kits

  • Confirm BSG presence using Western blot with anti-BSG antibodies

  • Quantify BSG levels in EVs from different cell types or disease states

• Functional studies:

  • Use BSG antibodies to neutralize EV function in recipient cells

  • Employ BSG knockout/knockdown approaches to assess its requirement for EV production

  • Track BSG-containing EVs with fluorescently labeled antibodies

• Imaging approaches:

  • Immunogold electron microscopy for precise localization of BSG on EVs

  • Super-resolution microscopy to visualize BSG clustering during EV biogenesis

  • Live cell imaging to track BSG dynamics during EV release and uptake

• Recipient cell analysis:

  • Examine effects of BSG-containing EVs on target cell phenotypes

  • Block BSG-mediated EV uptake using specific antibodies

  • Investigate signaling pathways activated by BSG-containing EVs