CEP95 Antibody

What is CEP95 and what cellular functions does it perform?

CEP95 is a centrosomal protein with a molecular weight of approximately 95 kDa . Its gene ontology annotations include protein binding and cytoskeleton functions . The protein is encoded by the human gene located at UniProt ID Q96GE4 (human), Q8BVV7 (mouse), and Q5XI03 (rat) . While specific functions are still being elucidated, its centrosomal localization suggests roles in cell division, microtubule organization, and centrosome-related processes .

What are the primary applications for CEP95 antibodies in research?

CEP95 antibodies are primarily utilized in the following applications:

• Western Blot (WB): Used to detect CEP95 protein expression levels in cell or tissue lysates. Most commercial antibodies can detect a band at approximately 95 kDa .

• Immunohistochemistry (IHC-P): Applied to paraffin-embedded tissues to visualize the spatial distribution of CEP95 in various tissues and tumors .

• Immunofluorescence: Some antibodies have been characterized for immunofluorescence applications to examine subcellular localization .

What species cross-reactivity should be expected with CEP95 antibodies?

Most commercial CEP95 antibodies demonstrate reactivity with human, mouse, and rat samples . When selecting an antibody for your research, verify the documented cross-reactivity in the product information. For example:

• Abcam's ab230305 reacts with mouse and human samples

• Boster Bio's A14760-1 reacts with human, mouse, and rat samples

• Sigma's HPA052426 is primarily validated with human samples

Cross-reactivity should be experimentally validated if working with species not explicitly listed in the manufacturer's documentation.

How should I validate CEP95 antibody specificity for my experimental system?

Proper validation requires multiple approaches:

• Positive control selection: Use tissues or cell lines known to express CEP95. According to product documentation, mouse heart tissue lysate serves as an effective positive control for Western blot applications . For IHC-P, human bladder and colon cancer tissues have been validated as positive controls .

• Molecular weight verification: CEP95 should show a primary band at approximately 95 kDa in Western blot applications, though some isoforms may produce additional bands at 77 kDa .

• Knockdown validation: For definitive specificity confirmation, compare antibody staining between wild-type cells and those with CEP95 knockdown or knockout. Consider using available CEP95 ORF clones for overexpression controls .

• Multiple antibody comparison: Use antibodies targeting different epitopes of CEP95 to confirm specificity. Commercial antibodies target different regions - for example, Abcam's ab230305 targets amino acids 550-850 , while Sigma's HPA061371 targets a different peptide sequence .

What are the optimal sample preparation methods for CEP95 detection?

Sample preparation methods vary by application:

For Western Blot:

• Use lysis buffers containing protease inhibitors to prevent degradation

• Heat samples at 95°C for 5 minutes in loading buffer containing SDS and DTT

• Load 20-50 µg of total protein per lane

• Use recommended antibody dilutions (typically 1/1000-1/5000 for WB)

For IHC-P:

• Fix tissues in 10% neutral buffered formalin

• Perform antigen retrieval (specific methods may vary by antibody)

• Block endogenous peroxidase activity

• Use recommended antibody dilutions (typically 1/20-1/500 for IHC-P)

How can I optimize CEP95 antibody performance in challenging experimental conditions?

For improved antibody performance:

• Titrate antibody concentrations: Test a range of dilutions to find optimal signal-to-noise ratio

• Modify blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) at various concentrations

• Adjust incubation parameters: Try different incubation times and temperatures

• Sample preprocessing: Consider sample enrichment techniques if detecting low-abundance targets

• Signal amplification: For weaker signals, explore biotin-streptavidin amplification systems or higher-sensitivity substrates

What are common issues when using CEP95 antibodies in Western blotting and how can they be resolved?

What factors should be considered when interpreting CEP95 expression patterns in disease models?

When examining CEP95 expression in disease models:

• Baseline expression: Understand normal CEP95 expression in relevant tissues. The Human Protein Atlas provides tissue-specific expression data .

• Context-specific regulation: Expression patterns may differ between in vitro and in vivo systems. For example, CEP95 expression might be altered in cell culture models compared to primary tissues .

• Relationship to disease markers: Consider correlating CEP95 expression with established disease markers. Some studies have investigated centrosomal proteins in cancer progression .

• Technical considerations: Different fixation methods, antibody clones, and detection systems can influence staining patterns. Compare results across multiple methodologies when possible .

• Quantification methods: Apply appropriate quantification techniques (densitometry for WB, digital image analysis for IHC) with suitable statistical analysis .

How can I co-localize CEP95 with other centrosomal markers in immunofluorescence studies?

For successful co-localization experiments:

• Antibody compatibility: Select CEP95 antibodies and other centrosomal marker antibodies from different host species to avoid cross-reactivity

• Sequential staining protocol:

  • Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.2% Triton X-100 (5 minutes)

  • Block with 3% BSA in PBS (30 minutes)

  • Apply primary antibodies sequentially (if from same species) or simultaneously (if from different species)

  • Use fluorophore-conjugated secondary antibodies with non-overlapping emission spectra

  • Include DAPI for nuclear counterstaining

• Controls: Include single-stain controls to assess bleed-through and secondary-only controls to assess non-specific binding

• Imaging parameters: Use confocal microscopy with appropriate resolution settings to visualize centrosomal structures, which are typically 1-2 μm in diameter

How do different commercial CEP95 antibodies compare in terms of applications and performance?

What considerations should guide antibody selection for different experimental objectives?

Selection criteria vary based on research goals:

• For protein expression studies (Western blot):

  • Select antibodies validated specifically for WB applications

  • Consider antibodies targeting conserved regions if studying across multiple species

  • Abcam's ab230305 and Boster's A14760-1 show strong validation for WB

• For localization studies (IHC/IF):

  • Choose antibodies specifically validated for microscopy applications

  • Consider the subcellular localization data provided by manufacturers

  • Sigma's HPA052426 and HPA061371 have extensive tissue validation through the Human Protein Atlas

• For functional studies:

  • Select antibodies targeting functional domains if the goal is to disrupt protein function

  • Consider whether the antibody recognizes denatured or native forms of the protein

• For developmental or comparative studies:

  • Prioritize antibodies with known cross-reactivity to species of interest

  • Verify epitope conservation across species through sequence alignment