prom1a Antibody

What is PROM1 and why are antibodies against it important in research?

PROM1 (Prominin-1, also known as CD133 or AC133) is a pentaspan transmembrane glycoprotein that plays a critical role in the maintenance of primary cilia. The protein is expressed in hematopoietic stem cells, endothelial progenitor cells, neuronal and glial stem cells, as well as in adult tissues including kidney, mammary glands, trachea, salivary glands, placenta, digestive tract, and testes. PROM1 has significant research importance as it serves as a key marker for hematopoietic stem cells in transplant models and has been identified as a marker of tumor-initiating cells . Antibodies against PROM1 are essential tools for studying its expression patterns, protein interactions, and functional roles in normal development and disease conditions, particularly in retinal degeneration models where PROM1 mutations have been implicated .

What are the common antibodies used for detecting PROM1?

Several antibodies have been developed for detecting PROM1 across different species. For mouse PROM1, the rat monoclonal antibody mAB 13A4 is the most commonly used reagent with over 300 academic publications citing its use . Another antibody frequently used is the mouse monoclonal antibody ab27699. For human CD133/PROM1, antibody clone 170411 (catalog MAB11331) is available for detection of the human protein . It's important to note that PROM1 antibodies tend to be species-specific due to relatively low conservation of primary amino acid sequence between species (for example, human and mouse PROM1 proteins share only approximately 61% sequence identity) .

How can I verify the specificity of a PROM1 antibody?

Verifying antibody specificity is crucial for interpreting experimental results. One recommended approach is to perform western blot analysis comparing wild-type tissue with tissue from PROM1 knockout models. Research has demonstrated that both mAB 13A4 and ab27699 recognize a protein just over 100 kDa in size in wild-type mouse retina that is absent in extracts from Prom1 rd19 knockout retina . Additionally, immunoprecipitation assays can be used to confirm antibody specificity. This involves homogenizing tissue in radioimmunoprecipitation assay (RIPA) buffer, incubating with the antibody, isolating the antibody-protein complex using Protein A agarose beads, and then analyzing the immunoprecipitated fraction by western blot with a second antibody recognizing a different epitope of the same protein .

Why might different antibodies against PROM1 produce discrepant results?

Discrepancies in protein detection between different PROM1 antibodies can arise from several factors, primarily differences in epitope recognition. A significant example is the discrepancy observed between mAB 13A4 and ab27699 when measuring PROM1 protein levels in postnatal mouse retina. When measured with mAB 13A4, PROM1 protein levels peaked at postnatal day 8, while ab27699 detected peak levels five days later at postnatal day 13 . Moreover, mAB 13A4 showed approximately three to five-fold lower levels of PROM1 at postnatal days 13 and beyond compared to ab27699. Statistical analysis using two-way ANOVA confirmed significant effects of both developmental stage and antibody used on the measured PROM1 protein levels .

The observed discrepancy is attributed to mAB 13A4 recognizing a structural epitope that is affected by the inclusion of a photoreceptor-specific alternative exon (exon 19a) in the third extracellular domain of PROM1. This tissue-specific splicing event reduces the affinity of mAB 13A4 to the photoreceptor PROM1 isoform, causing underestimation of PROM1 levels compared to ab27699 . This case illustrates the importance of understanding antibody epitopes when interpreting protein expression data, especially in tissues with isoform diversity.

How does alternative splicing of PROM1 affect antibody recognition?

Alternative splicing generates multiple PROM1 isoforms that can significantly impact antibody recognition. The most thoroughly documented example involves the photoreceptor-specific isoform (SV8) which includes alternative exon 19a in the third extracellular domain. Research has demonstrated that inclusion of this exon reduces the affinity of mAB 13A4 for PROM1 by disrupting the structural epitope recognized by this antibody .

Interestingly, deletion mutagenesis experiments revealed that removing six amino acids adjacent to exon 19a in the photoreceptor-specific PROM1 isoform restored mAB 13A4 binding. This finding supports the model that mAB 13A4 recognizes a conformational epitope dependent on the precise three-dimensional arrangement of the protein's extracellular domains . When planning experiments with PROM1 antibodies, researchers should consider which isoforms are expected in their tissue of interest and select antibodies accordingly to avoid misinterpretation of expression patterns.

What is known about the mAB 13A4 epitope and how was it mapped?

The epitope of mAB 13A4 was investigated through systematic deletion mutagenesis of the PROM1 protein. These studies revealed that mAB 13A4 recognizes a conformational epitope rather than a linear sequence of amino acids. Deletions in both the second and third extracellular domains of PROM1 disrupted the mAB 13A4 epitope, indicating that these regions contribute to forming the three-dimensional structure recognized by the antibody .

The complex nature of this epitope is further evidenced by the observation that mutations hundreds of amino acids apart could abolish mAB 13A4 binding. Computational modeling of PROM1's three-dimensional structure guided the design of additional mutations to test the structural hypothesis. Three specific mutations were created:

• A deletion of six amino acids adjacent to exon 19a in the photoreceptor-specific isoform, which restored mAB 13A4 binding

• A deletion of 15 amino acids (D-7) in the third extracellular domain opposite to another deletion (D+4), which retained the mAB 13A4 epitope

• A deletion of 15 amino acids (Del EC 2) in the upper half of the second extracellular domain, which resulted in loss of the mAB 13A4 epitope

The effects of these mutations aligned with predictions based on the computational model, providing empirical evidence for the validity of the predicted PROM1 structure and confirming the conformational nature of the mAB 13A4 epitope.

What protein extraction methods are recommended for PROM1 antibody western blotting?

For optimal western blot detection of PROM1, tissues or cells should be lysed in RIPA buffer (50 mM Tris HCl-pH 8.0, 150 mM NaCl, 1.0% TritonX-100, 0.5% Sodium Deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktails. After homogenization, samples should be incubated on ice for 10 minutes and then cleared by centrifugation for 15 minutes . Typically, 20 μg of protein extract is sufficient for resolving on a 4-20% polyacrylamide SDS-PAGE gel.

When working with PROM1, it's important to note that it is a glycoprotein, and deglycosylation can affect antibody recognition. Studies have shown that mAB 13A4 can recognize deglycosylated PROM1, but with reduced signal intensity, sometimes resulting in inconsistent detection of certain isoforms . Therefore, when comparing PROM1 levels across different conditions or tissues, consistent sample preparation methods are crucial.

How can I detect PROM1 in different cellular compartments?

PROM1 is primarily localized to plasma membrane protrusions and is particularly enriched in primary cilia. For detecting PROM1 in different cellular compartments, immunofluorescence microscopy using specific antibodies is the recommended approach. When performing immunofluorescence studies, standard fixation protocols using 4% paraformaldehyde are generally suitable.

For subcellular fractionation studies, it's important to consider that PROM1 can form higher-order complexes under native conditions, which may affect its extraction and detection . Different detergents have varying abilities to solubilize PROM1 from different membrane domains, so optimization may be required depending on the specific cellular compartment being investigated.

What controls should be included when using PROM1 antibodies in immunohistochemistry?

When performing immunohistochemistry with PROM1 antibodies, several controls are essential:

• Negative tissue control: Include tissues from PROM1 knockout models (such as Prom1 rd19 mice) to confirm antibody specificity .

• Blocking peptide control: When available, pre-incubation of the antibody with its specific antigenic peptide should abolish specific staining.

• Secondary antibody control: Omit the primary antibody to assess non-specific binding of the secondary antibody.

• Positive control tissues: Include tissues known to express PROM1, such as kidney or neuroepithelium where PROM1 was originally identified .

• Isoform considerations: For tissues known to express specific PROM1 isoforms (such as retina with the photoreceptor-specific isoform), consider using multiple antibodies recognizing different epitopes to ensure complete detection of all isoforms .

How should developmental expression data for PROM1 be interpreted?

The interpretation of PROM1 developmental expression data requires careful consideration of the antibodies used and the tissue-specific isoforms present. As shown in the table below, significant discrepancies can exist between different antibodies at specific developmental stages:

Data approximated from figures in references and

This discrepancy is attributed to changes in PROM1 isoform composition during retinal development. As photoreceptors mature, the expression of the photoreceptor-specific isoform (SV8) increases, which includes exon 19a that disrupts the mAB 13A4 epitope. Consequently, mAB 13A4 underestimates PROM1 levels in mature retina by approximately five-fold compared to ab27699 .

When interpreting developmental expression data, researchers should use multiple antibodies recognizing different epitopes when possible, and correlate protein detection with mRNA expression analysis of specific isoforms.

What factors can lead to false-negative or reduced PROM1 detection?

Several factors can contribute to false-negative or reduced PROM1 detection:

• Isoform-specific epitope masking: As demonstrated with mAB 13A4, tissue-specific alternative splicing can disrupt antibody epitopes. The inclusion of exon 19a in the photoreceptor-specific isoform reduces mAB 13A4 affinity, leading to underestimation of PROM1 levels .

• Protein glycosylation status: PROM1 is a glycoprotein, and changes in glycosylation can affect antibody recognition. Deglycosylated PROM1 was recognized by mAB 13A4 with reduced signal intensity .

• Protein denaturation conditions: Since mAB 13A4 recognizes a conformational epitope, excessive denaturation can reduce antibody binding. Optimal SDS concentration and heating conditions should be empirically determined.

• Epitope masking by protein interactions: Under native conditions, PROM1 can form higher-order complexes that might mask certain epitopes .

• Species cross-reactivity limitations: Due to the relatively low sequence conservation between species (61% identity between human and mouse), antibodies are typically species-specific. Using an antibody developed against one species to detect PROM1 in another species may result in false negatives .

How can contradictory results from different PROM1 antibodies be resolved?

When facing contradictory results from different PROM1 antibodies, consider the following approaches:

• Characterize the epitopes: If possible, determine which domains or regions of PROM1 are recognized by each antibody. For instance, mAB 13A4 recognizes a structural epitope involving the second and third extracellular domains .

• Consider isoform expression: Determine which PROM1 isoforms are expressed in your experimental system. If photoreceptor-specific or other alternatively spliced isoforms are present, certain antibodies may exhibit reduced affinity .

• Validate with genetic models: Use PROM1 knockout or mutant models as negative controls to confirm antibody specificity .

• Employ complementary techniques: Correlate protein detection with mRNA expression analysis using isoform-specific primers.

• Use multiple antibodies: When possible, use multiple antibodies targeting different epitopes and report results from all antibodies tested.

• Create epitope-tagged constructs: For overexpression studies, adding epitope tags can provide consistent detection independent of PROM1 structural variations.

How can mAB 13A4 be used to evaluate PROM1 structure?

The discovery that mAB 13A4 recognizes a structural epitope makes it a valuable tool for evaluating the effect of mutations on PROM1 structure. Since the epitope is disrupted by changes in the three-dimensional arrangement of PROM1's extracellular domains, mAB 13A4 binding can serve as an indicator of proper protein folding .

This application was demonstrated through deletion mutagenesis experiments where mutations hundreds of amino acids apart abolished mAB 13A4 binding. The results of these experiments aligned with a computationally predicted helical bundle structure of PROM1, providing empirical evidence for its validity . Researchers investigating novel PROM1 mutations, particularly those associated with retinal degeneration, could use mAB 13A4 binding as a preliminary indicator of structural perturbation before proceeding to more complex structural analyses.

What are the considerations for studying PROM1 in disease models?

When studying PROM1 in disease models, several key considerations should be addressed:

• Mutation effects on antibody binding: Disease-causing mutations may alter the PROM1 structure, potentially affecting antibody recognition. For instance, a truncating mutation in the third extracellular domain abolished mAB 13A4 binding .

• Isoform-specific effects: Mutations may affect specific PROM1 isoforms differently. The photoreceptor-specific isoform (SV8) has distinct structural properties from other isoforms, which could influence how mutations impact protein function .

• Higher-order complex formation: Under native conditions, PROM1 can form higher-order complexes. Mutations may affect these interactions, potentially contributing to disease mechanisms. This is particularly relevant for dominant mutations in cone-rod dystrophy .

• Antibody selection: For comprehensive PROM1 detection in disease models, select antibodies that recognize epitopes unlikely to be affected by the mutation of interest. When studying retinal degeneration models, consider that mAB 13A4 may underestimate PROM1 levels in photoreceptors .