CEP97 Antibody, FITC conjugated

What is CEP97 and what cellular functions does it regulate?

CEP97 (Centrosomal protein of 97 kDa), also known as LRRIQ2 (Leucine-rich repeat and IQ domain-containing protein 2), is a centrosomal protein involved in centriole length regulation and ciliogenesis. CEP97 contains one IQ domain, eight leucine-rich repeats, and one LRRCT domain. It functions primarily at the centrosome where it collaborates with other centrosomal proteins to regulate centriole structure and function. The protein plays a critical role in preventing aberrant cilia formation by capping the distal end of centrioles, functioning as a gatekeeper for primary cilium assembly. CEP97 has a calculated molecular weight of approximately 97 kDa, though observed weights may vary between 72-140 kDa in different experimental conditions .

How does FITC conjugation affect CEP97 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent labeling of the CEP97 antibody, eliminating the need for secondary antibodies in fluorescence-based applications. This conjugation allows for direct visualization of CEP97 in immunofluorescence, flow cytometry, and confocal microscopy experiments. The FITC fluorophore has an excitation maximum at approximately 495 nm and emission maximum at about 519 nm, producing a green fluorescence. Unlike unconjugated antibodies, FITC-conjugated CEP97 antibodies require special storage conditions, including protection from light exposure and storage at 2-8°C without freezing to maintain optimal fluorescence intensity . The conjugation process is optimized to ensure that antibody binding specificity and affinity for CEP97 are preserved while providing strong fluorescent signal.

What is the optimal storage protocol for maintaining FITC-CEP97 antibody activity?

For maximum preservation of both antibody activity and fluorescence intensity, FITC-conjugated CEP97 antibodies should be stored at 2-8°C for up to twelve months without detectable loss of activity. The antibody should be protected from prolonged exposure to light to prevent photobleaching of the FITC fluorophore. Unlike unconjugated antibodies, FITC-conjugated antibodies should not be frozen as this can lead to denaturation and fluorescence quenching. For long-term storage, the antibody should be kept in its original buffer containing stabilizing components such as glycerol, BSA, and sodium azide. If aliquoting is necessary, use sterile tubes and minimize freeze-thaw cycles. Before each use, allow the antibody to reach room temperature and centrifuge briefly to collect the solution at the bottom of the vial .

What species reactivity is expected for CEP97 antibodies?

Based on available data, CEP97 antibodies show confirmed reactivity with human and mouse samples, with some evidence for canine reactivity. Rabbit polyclonal antibodies against CEP97 have been validated to recognize the protein in various human cell lines including HEK-293, HeLa, HL-60, and PC-3 cells, as well as in mouse systems. For canine samples, reactivity has been observed in MDCK cells. When considering cross-reactivity for unvalidated species, sequence homology analysis should be performed, as CEP97 is relatively conserved across mammalian species. In particular, human CEP97 shares high sequence similarity with primate, bovine, and rodent orthologs, suggesting possible cross-reactivity with these species .

How can CEP97 antibody be validated for specificity in centrosome research?

Rigorous validation of CEP97 antibody specificity for centrosome research requires multiple orthogonal approaches. First, perform Western blot analysis using positive control lysates from cell lines known to express CEP97 (such as HeLa, HEK-293, and RAW264.7 cells) alongside negative controls. A specific antibody should detect a band at the expected molecular weight (72-140 kDa, depending on post-translational modifications). Second, conduct peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish signal in Western blot and immunostaining applications, as demonstrated with CEP97 antibody on RAW264.7 cell lysates . Third, perform immunofluorescence co-localization studies with established centrosomal markers such as γ-tubulin or pericentrin. Fourth, use siRNA or CRISPR knockout/knockdown approaches to deplete CEP97 and confirm diminished antibody signal. Finally, conduct immunoprecipitation followed by mass spectrometry to verify that the antibody captures the correct protein target .

What are the critical differences between CEP97 detection in mitotic versus interphase cells?

Detecting CEP97 with FITC-conjugated antibodies reveals distinct localization patterns and expression levels throughout the cell cycle. In interphase cells, CEP97 primarily localizes to the mother centriole of the centrosome, forming a cap-like structure at the distal end that prevents aberrant cilia formation. The signal appears as one or two distinct puncta near the nucleus, depending on centrosome duplication status. In mitotic cells, CEP97 shows dynamic redistribution, with enrichment at the spindle poles and potential changes in abundance. The fluorescence intensity of CEP97 staining may increase during S phase as centrosomes duplicate, followed by a different pattern during mitosis when centrioles separate. When imaging, it's crucial to co-stain with cell cycle markers (e.g., phospho-histone H3 for mitosis) and additional centrosomal proteins to accurately interpret CEP97 dynamics. Microscopy settings must be optimized differently for mitotic cells, which may require shorter exposure times to prevent oversaturation due to the concentrated centrosomal signal at spindle poles .

How do post-translational modifications affect CEP97 antibody recognition?

Post-translational modifications (PTMs) of CEP97 can significantly impact antibody recognition, particularly for antibodies targeting modified epitopes. CEP97 undergoes several PTMs including phosphorylation, ubiquitination, and possibly SUMOylation that regulate its function and localization throughout the cell cycle. These modifications can mask or expose epitopes, altering antibody binding efficiency. The observed molecular weight discrepancy between calculated (97 kDa) and detected CEP97 (130-140 kDa in some studies) suggests extensive PTMs or alternatively spliced isoforms . When selecting a CEP97 antibody, researchers should consider whether the immunogen sequence (e.g., amino acids 581-630 in human CEP97) contains known modification sites. For comprehensive analysis, it's advisable to use antibodies recognizing different CEP97 epitopes or specific phospho-antibodies when studying cell cycle-dependent functions. Treating samples with phosphatases or deubiquitinating enzymes before immunostaining can help determine if PTMs affect antibody recognition in your experimental system .

What is the optimal protocol for CEP97 detection in ciliated versus non-ciliated cells?

Detecting CEP97 requires different protocols for ciliated versus non-ciliated cells due to its dynamic localization during ciliogenesis. For non-ciliated cells (proliferating cells), standard fixation with 4% paraformaldehyde for 15 minutes at room temperature preserves centrosomal structure while maintaining FITC fluorescence. In these cells, CEP97 is prominently localized at the mother centriole. For ciliated cells (typically serum-starved), cold methanol fixation (-20°C for 10 minutes) may better preserve ciliary and centrosomal structures. In ciliated cells, CEP97 is notably absent from the mother centriole that has converted to a basal body, making co-staining with ciliary markers (such as acetylated tubulin) essential for proper interpretation.

The staining protocol should include:

• Permeabilization with 0.1-0.5% Triton X-100 (adjust concentration based on cell type)

• Blocking with 3-5% BSA or normal serum

• FITC-CEP97 antibody incubation at optimal dilution (typically 1:50-1:200) for 1-2 hours at room temperature or overnight at 4°C

• Co-staining with basal body/ciliary markers using fluorophores with non-overlapping spectra

• Counterstaining of DNA with DAPI

For simultaneous visualization of CEP97 at different stages of ciliogenesis, mixed populations of ciliated and non-ciliated cells can be analyzed by manipulating culture conditions to induce partial ciliation .

What dilutions and controls are recommended for flow cytometry using FITC-CEP97 antibody?

For optimal flow cytometry experiments with FITC-conjugated CEP97 antibody, begin with a titration experiment using the dilution ranges above. While CEP97 is primarily an intracellular protein localized to centrosomes, proper permeabilization is critical. Use a standard intracellular staining protocol:

• Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS

• Block with 3% BSA in permeabilization buffer

• Stain with FITC-CEP97 antibody at determined optimal concentration

• Wash thoroughly to remove unbound antibody

Essential controls include:

• FITC-conjugated isotype control matched to your antibody (rabbit IgG-FITC)

• Fluorescence Minus One (FMO) controls

• Cells with known CEP97 expression levels (positive control)

• CEP97-depleted cells (negative control)

To assess specificity, pre-incubate an aliquot of antibody with blocking peptide representing the immunogen sequence (amino acids 581-630) before cell staining. Signal compensation is crucial when multiplexing with other fluorophores .

How should sample preparation differ for various CEP97 detection methods?

For Western blot analysis, prepare lysates in RIPA buffer supplemented with protease inhibitors. When analyzing centrosomal proteins by immunofluorescence, pre-extraction with 0.5% Triton X-100 in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9) for 30 seconds before fixation can reduce cytoplasmic background and enhance centrosomal signal. For IHC applications, antigen retrieval is critical - use Tris-EDTA buffer (pH 9.0) as recommended in the validation data. For all applications, freshly prepared samples yield optimal results, though fixed cells can be stored at 4°C in PBS containing 0.02% sodium azide for up to one week before antibody staining .

How can non-specific binding of FITC-CEP97 antibody be minimized?

Non-specific binding of FITC-conjugated CEP97 antibody can manifest as diffuse cytoplasmic staining or unexpected nuclear signals. To minimize this issue, implement a comprehensive optimization strategy. First, increase blocking stringency by using 5-10% normal serum matched to the host species of your secondary antibodies, combined with 1% BSA and 0.1% Tween-20. For particularly problematic samples, add 10% normal serum from the species your cells originate from. Second, optimize antibody concentration through careful titration; for FITC-conjugated antibodies, higher concentrations often increase background rather than specific signal. Third, include 0.1-0.3M NaCl in your antibody dilution buffer to reduce ionic interactions. Fourth, extend washing steps (4-5 washes of 5 minutes each) with PBS containing 0.05-0.1% Tween-20. Fifth, pre-absorb the antibody with acetone powder prepared from a cell line that doesn't express CEP97. Finally, prepare cells with a pre-extraction step (0.5% Triton X-100 in PHEM buffer for 30 seconds before fixation) to remove soluble cytoplasmic proteins while retaining centrosome-bound CEP97 .

What are the potential causes of diminished FITC signal when detecting CEP97?

Diminished FITC signal when detecting CEP97 can result from multiple factors throughout the experimental workflow. The most common causes include:

• Photobleaching - FITC is particularly susceptible to photobleaching, so minimize exposure to light during all steps from storage to imaging. Use anti-fade mounting media containing agents like p-phenylenediamine or commercial products like ProLong Gold

• Suboptimal fixation - Overfixation with paraformaldehyde can mask epitopes; limit fixation to 10-15 minutes at room temperature

• Inadequate permeabilization - CEP97 is located in centrosomes, which may require stronger permeabilization; optimize detergent concentration

• Degraded antibody - FITC-conjugated antibodies typically have shorter shelf lives than unconjugated ones; avoid freeze-thaw cycles

• Cell cycle-dependent expression - CEP97 levels and localization change throughout the cell cycle; synchronize cells if consistent detection is required

• Protein degradation during sample preparation - Always use freshly prepared protease inhibitors

• Competitive binding from endogenous biotin - Include an avidin/biotin blocking step if your system includes a biotin-streptavidin component

• Suboptimal imaging settings - Adjust microscope settings specifically for FITC fluorescence (excitation ~495nm, emission ~519nm)

Incorporating appropriate controls (e.g., a separate sample stained with a known working FITC-conjugated antibody) can help identify which stage of the protocol is problematic .

How can centrosomal CEP97 signal be distinguished from non-specific aggregates?

Distinguishing genuine centrosomal CEP97 signal from non-specific aggregates requires multiple validation approaches. True centrosomal CEP97 staining will appear as one or two distinct puncta per cell (depending on cell cycle stage), measuring approximately 0.5-1.0 μm in diameter with well-defined boundaries. These puncta will consistently co-localize with established centrosome markers like γ-tubulin or pericentrin in dual-labeling experiments. In contrast, non-specific aggregates typically show irregular morphology, variable size, random cellular distribution, and lack consistent co-localization with centrosomal markers.

To conclusively differentiate specific signal:

• Always perform co-staining with a centrosome marker using a spectrally distinct fluorophore

• Analyze multiple fields and calculate the percentage of cells showing co-localization

• Compare signal patterns between known positive controls (e.g., proliferating cells) and negative controls (e.g., fully ciliated cells where CEP97 should be absent from basal bodies)

• Examine the expected cell cycle dependence of CEP97 localization

• Perform z-stack imaging to ensure three-dimensional co-localization

• Use super-resolution microscopy techniques (SIM, STED, or STORM) to precisely define spatial relationship between CEP97 and other centrosomal proteins

• Validate with orthogonal approaches such as proximity ligation assays

These approaches will help ensure that the observed signal represents authentic CEP97 localization rather than antibody aggregates or non-specific binding .

How does CEP97 antibody staining pattern correlate with ciliogenesis stages?

CEP97 antibody staining patterns directly correlate with distinct stages of ciliogenesis, making it a valuable marker for tracking this process. In proliferating cells that do not form primary cilia, CEP97 shows strong localization to the distal end of mother centrioles, appearing as distinct puncta that partially co-localize with distal appendage proteins. As cells enter ciliogenesis upon serum starvation or cell cycle exit, CEP97 undergoes a characteristic displacement from the mother centriole, which is a prerequisite for basal body formation and subsequent axoneme extension.

The temporal sequence can be characterized by four distinct staining patterns:

• Pre-ciliation stage: Strong CEP97 signal at both mother and daughter centrioles

• Early ciliogenesis: Diminished CEP97 at the mother centriole but retained at daughter centriole

• Active ciliogenesis: Complete loss of CEP97 from mother centriole (now converted to basal body), maintaining signal only at daughter centriole

• Fully ciliated state: CEP97 absent from basal body but still present at daughter centriole

Quantitative analysis of CEP97 intensity at the mother centriole/basal body relative to ciliary markers (such as acetylated tubulin or Arl13b) provides a precise method to classify individual cells within a population according to their ciliogenesis status. This approach is particularly valuable for investigating factors that regulate the initiation of primary cilium formation .

What is the significance of the molecular weight discrepancy between predicted and observed CEP97?

The notable discrepancy between the predicted molecular weight of CEP97 (97 kDa) and its observed molecular weight on immunoblots (72 kDa in some studies, 130-140 kDa in others) reflects important biological properties of this protein. This variance can be attributed to several factors with significant research implications:

• Post-translational modifications: CEP97 undergoes extensive phosphorylation, particularly during mitosis, which can substantially increase its apparent molecular weight. The higher observed weight (130-140 kDa) likely represents heavily phosphorylated forms, while faster-migrating bands may represent dephosphorylated variants

• Alternative splicing: Multiple CEP97 isoforms have been documented, with different exon usage potentially contributing to size variations

• Protein structure: CEP97 contains leucine-rich repeats that can affect SDS binding and alter migration patterns during electrophoresis

• Proteolytic processing: Cell type-specific proteases may generate truncated versions of CEP97 with distinct functional properties

• Detection method sensitivity: The epitope recognized by specific antibodies may be differentially accessible in various protein conformations or modified states

Researchers should carefully document which form they detect in their experimental system and consider using phosphatase treatment of lysates to determine if the higher molecular weight forms are due to phosphorylation. For comprehensive analysis, use multiple antibodies targeting different regions of CEP97 to capture the complete profile of protein variants .

How can FITC-CEP97 antibody be used in live-cell imaging experiments?

While conventional FITC-conjugated antibodies cannot penetrate intact cell membranes for live-cell imaging, advanced approaches enable dynamic visualization of CEP97 in living cells. For such applications, researchers must employ specialized delivery methods:

• Cell-penetrating peptide conjugation: FITC-CEP97 antibodies can be conjugated to cell-penetrating peptides (CPPs) like TAT or Penetratin to facilitate cellular uptake while maintaining fluorescence and binding specificity

• Electroporation: Gentle electroporation protocols allow antibody delivery with minimal cellular damage; optimize voltage and pulse duration for your specific cell type

• Microinjection: For precise temporal control, direct microinjection of FITC-CEP97 antibody into individual cells allows immediate imaging post-injection

• Transient cell permeabilization: Brief treatment with streptolysin O creates temporary pores for antibody entry, followed by resealing in calcium-containing media

Once delivered, live-cell imaging should employ minimal laser power and interval acquisition to reduce photobleaching and phototoxicity. For optimal visualization, use spinning disk confocal microscopy or lattice light-sheet microscopy with environmental control (37°C, 5% CO2). To track CEP97 dynamics through cell cycle progression, combine with far-red fluorescent probes marking cell cycle stages. Note that antibody binding may potentially interfere with CEP97 function or interactions, so validation against fixed-cell observations is essential .