PROM1 Monoclonal Antibody

What is PROM1 and why is it significant in research?

PROM1 (also known as CD133 or AC133) is a glycosylated membrane protein with five transmembrane domains and three extracellular domains. It belongs to a conserved family of proteins that modulate the architecture of cellular protrusions such as microvilli and cilia . PROM1 plays essential roles in maintaining primary cilia, and mutations in the Prom1 gene have been associated with various types of retinal degeneration, primarily cone-rod dystrophy . The protein is particularly significant in developmental biology, stem cell research, and ophthalmology due to its role in cellular architecture and its connection to retinal disorders. Mouse models lacking Prom1 or expressing dominant mutations like Arg373Cys recapitulate retinal degeneration phenotypes and display defects in disk morphogenesis, making this protein a critical research target .

What are the known isoforms of PROM1 and how do they affect antibody recognition?

The Prom1 gene produces multiple splicing isoforms that can be tissue and cell-type specific. In mouse, six alternative exons can potentially produce 24 splice variants, although only eight have been enumerated to date . The SV8 isoform, which is photoreceptor-specific, includes microexon 19a that introduces 6 amino acids in the third extracellular domain . This structural variation significantly affects the recognition of PROM1 by antibodies, particularly mAB 13A4, which shows approximately three to five-fold lower affinity for the photoreceptor-specific isoform compared to other variants . When conducting experiments with tissues expressing multiple PROM1 isoforms, researchers must consider how antibody affinity varies across these variants to avoid misinterpreting expression data.

What methods are used to detect PROM1 in experimental settings?

PROM1 detection typically relies on immunological methods using specific antibodies. For mouse PROM1, the rat monoclonal antibody mAB 13A4 is the primary reagent . Other antibodies, such as the mouse monoclonal ab27699, are also used . Detection methods include:

• Western blotting: Typically performed under denaturing conditions using RIPA buffer for protein extraction

• Immunohistochemistry/immunofluorescence: For tissue localization studies

• Flow cytometry: Often used in stem cell applications

When selecting a detection method, researchers should consider the nature of the epitope (linear vs. structural) and the specific isoforms present in their experimental system. For instance, when analyzing photoreceptor cells, the reduced affinity of mAB 13A4 for the SV8 isoform might necessitate alternative detection approaches or validation with multiple antibodies .

How does the structural epitope of mAB 13A4 impact experimental design and data interpretation?

mAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1, rather than a linear sequence of amino acids . This structural recognition has significant implications for experimental design:

• Protein preparation methods: Harsh denaturing conditions may disrupt the structural epitope, reducing antibody binding.

• Expression systems: When expressing recombinant PROM1, proper protein folding is critical for epitope presentation. The choice of expression system and purification methods should preserve the native protein structure.

• Mutation analysis: Since deletions in the second and third extracellular domains of PROM1 can disrupt the mAB 13A4 epitope, researchers studying PROM1 mutations should be aware that loss of antibody binding might not necessarily indicate loss of protein expression, but rather structural changes .

• Comparative studies: When comparing PROM1 levels across developmental stages or tissues, researchers must account for potential variations in antibody affinity due to differential expression of PROM1 isoforms .

The structural nature of the epitope makes mAB 13A4 a useful tool for evaluating the effect of mutations on PROM1 structure, as changes to the PROM1 sequence hundreds of amino acids apart can abolish the epitope .

What explains the discrepancy between PROM1 detection by different antibodies, and how should researchers address this issue?

A significant discrepancy has been observed between PROM1 protein levels measured in postnatal retina by mAB 13A4 and the mouse monoclonal antibody ab27699 . When measured by mAB 13A4, PROM1 protein levels peaked at postnatal day 8, whereas with ab27699, the peak was recorded five days later . Furthermore, mAB 13A4 showed approximately three to five-fold lower levels of PROM1 at postnatal days 13 and beyond compared to ab27699 .

This discrepancy is attributed to:

• Isoform specificity: mAB 13A4 has reduced affinity for the photoreceptor-specific SV8 isoform that becomes predominant in later developmental stages .

• Epitope accessibility: The structural epitope recognized by mAB 13A4 may become less accessible as PROM1 forms complexes or changes conformation during development.

To address these discrepancies, researchers should:

• Use multiple antibodies that recognize different epitopes of PROM1

• Validate antibody specificity using knockout controls (as demonstrated with the Prom1rd19 mutant retina)

• Consider the developmental timing and tissue-specific expression of PROM1 isoforms

• Include controls that account for potential variations in antibody affinity

• Correlate protein detection with gene expression data when possible

What experimental approaches are most effective for mapping structural epitopes of PROM1 antibodies?

The mapping of structural epitopes requires specialized approaches beyond those used for linear epitopes. Based on the study of mAB 13A4, effective approaches include:

• Tiled deletion mutagenesis: Creating a series of deletion mutants throughout the protein sequence to identify regions critical for antibody binding . This approach helped identify that deletions in both the second and third extracellular domains disrupted the mAB 13A4 epitope .

• Expression of splice variants: Comparing antibody binding to naturally occurring splice variants can reveal how alternative exons affect epitope presentation . This revealed that the inclusion of exon 19a in the photoreceptor-specific isoform reduced mAB 13A4 affinity .

• Structure-guided targeted mutations: Using computational models of protein structure to predict critical regions for antibody binding. The researchers used RobeTTa-predicted structures to design three targeted mutations that confirmed the structural nature of the mAB 13A4 epitope .

• Epitope recovery experiments: Strategic deletions adjacent to known problematic regions may sometimes restore antibody binding. For example, deletion of six amino acids adjacent to the alternative exon 19a restored the affinity of mAB 13A4 to the photoreceptor-specific PROM1 isoform .

• Computational structural analysis: Using tools like RobeTTa and AlphaFold to predict protein structures and identify potential structural epitopes based on surface accessibility and electrostatic properties .

How can alternative splicing of PROM1 be systematically analyzed to predict antibody recognition patterns?

Alternative splicing significantly impacts antibody recognition of PROM1, as demonstrated by the reduced affinity of mAB 13A4 for the photoreceptor-specific SV8 isoform . To systematically analyze how alternative splicing affects antibody recognition:

• Comprehensive isoform characterization: Identify all relevant splice variants in your experimental system using RNA-seq or targeted PCR approaches. For PROM1, six alternative exons can generate up to 24 potential splice variants, though only eight have been enumerated to date .

• Recombinant expression system: Express each isoform with epitope tags in cell culture systems (such as Neuro 2a cells) to enable detection independent of the antibody being evaluated .

• Comparative binding analysis: Assess antibody binding to each isoform using Western blotting, ELISA, or surface plasmon resonance to quantify relative affinities.

• Structural modeling: Use computational tools to predict how alternative exons might alter protein folding and epitope presentation. For instance, inclusion of exon 19a in PROM1 lengthens the second helix of the third extracellular domain, causing a kink in the helical bundle that likely disrupts the mAB 13A4 epitope .

• Domain swapping experiments: Create chimeric constructs exchanging domains between isoforms to pinpoint regions responsible for differential antibody recognition.

• Site-directed mutagenesis: Introduce point mutations in critical regions to assess their impact on antibody binding without dramatically altering protein structure.

What considerations are important when interpreting developmental expression data for PROM1 using mAB 13A4?

When using mAB 13A4 to evaluate PROM1 expression during development, researchers should consider:

• Isoform switching: Developmental processes often involve changes in isoform expression patterns. Since mAB 13A4 has reduced affinity for the photoreceptor-specific SV8 isoform, apparent decreases in PROM1 levels during retinal development may reflect isoform switching rather than actual protein reduction .

• Quantification accuracy: The five-fold underestimation of PROM1 levels by mAB 13A4 compared to ab27699 in mature retina highlights the potential for significant quantification errors . Researchers should validate developmental expression patterns with multiple antibodies or complementary techniques.

• Tissue-specific effects: The impact of isoform variation on antibody recognition is tissue-specific. The photoreceptor-specific exon 19a affects mAB 13A4 binding in retinal tissue but would not impact detection in tissues where this exon is not expressed .

• Temporal resolution: The timing of developmental events may appear shifted when using different antibodies. For instance, PROM1 protein levels peaked at postnatal day 8 when measured by mAB 13A4 but five days later when measured by ab27699 .

• Cross-validation strategies: To obtain accurate developmental expression data, researchers should:

  • Compare results from multiple antibodies recognizing different epitopes

  • Correlate protein detection with mRNA expression analysis

  • Use epitope-tagged PROM1 constructs in transgenic models when possible

  • Consider absolute quantification methods such as mass spectrometry for critical developmental timepoints

What protocols are recommended for validating PROM1 antibody specificity?

To ensure the specificity of PROM1 antibodies, researchers should follow these validation protocols:

• Genetic knockout controls: The most definitive validation approach is testing antibody reactivity in tissue from PROM1 knockout models. For instance, the study confirmed the specificity of both mAB 13A4 and ab27699 by showing they recognized a protein just over 100 kDa in wild-type retina that was absent in extract from Prom1rd19 knockout retina .

• Overexpression systems: Express epitope-tagged PROM1 variants in cell culture (e.g., Neuro 2a cells) to create positive controls with independent means of detection .

• Peptide competition assays: For antibodies targeting linear epitopes, pre-incubation with the immunizing peptide should abolish specific binding.

• Multiple antibody concordance: Compare detection patterns using multiple antibodies targeting different epitopes of PROM1.

• Correlation with mRNA expression: Verify that protein detection correlates with mRNA expression patterns across tissues and developmental stages.

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of the immunoprecipitated protein to ensure the antibody is capturing the intended target.

What are the optimal conditions for detecting PROM1 in Western blot applications?

Based on the protocols used in the analyzed research, optimal conditions for PROM1 detection by Western blot include:

• Sample preparation:

  • Flash-freeze tissue samples immediately after collection

  • Lyse samples in RIPA buffer (50 mM Tris HCl-pH 8.0) containing protease inhibitors

  • Process cultured cells 24 hours post-transfection for recombinant expression systems

• Protein separation:

  • Use SDS-PAGE gels capable of resolving proteins around 100 kDa effectively

  • Include positive controls (recombinant PROM1) and negative controls (knockout tissue) in each experiment

• Transfer and detection:

  • For structural epitopes like that of mAB 13A4, consider using mild transfer conditions to preserve protein folding when possible

  • Block membranes thoroughly to minimize background

  • When working with retinal samples, be aware that PROM1 appears just over 100 kDa in size

• Antibody selection and application:

  • For comprehensive analysis, use both mAB 13A4 and alternative antibodies like ab27699

  • Consider the specific isoforms present in your experimental system when interpreting band intensities

  • For tagged constructs, compare detection with both anti-PROM1 and anti-tag antibodies to assess epitope availability

• Data analysis:

  • Use appropriate normalization controls

  • When comparing PROM1 levels across developmental stages or tissues, account for potential variations in antibody affinity due to isoform differences

What are the key considerations for designing experiments involving PROM1 antibodies?

Based on the current understanding of PROM1 antibody characteristics, researchers should:

• Select appropriate antibodies: Choose antibodies based on the specific research question, considering the nature of the epitope and potential isoform specificity.

• Include proper controls: Always include positive and negative controls to validate antibody specificity in each experimental system.

• Consider isoform composition: Account for tissue-specific and developmental variations in PROM1 isoform expression when interpreting antibody-based detection results .

• Use multiple detection methods: Complement antibody-based detection with alternative approaches such as mRNA analysis or epitope tagging when possible.

• Understand epitope characteristics: Recognize that structural epitopes like that of mAB 13A4 may be influenced by mutations or splice variants that affect protein folding, even when distant from the binding site .

• Validate quantitative comparisons: When comparing PROM1 levels across conditions, use multiple antibodies to ensure consistent results, especially when analyzing tissues with variable isoform expression .