ccdc149a Antibody

What is CCDC149 protein and why is it studied?

CCDC149 (Coiled-Coil Domain Containing 149) is a human protein whose function remains largely uncharacterized in current research . It contains coiled-coil structural motifs, which typically mediate protein-protein interactions and are involved in diverse cellular processes including vesicular transport, protein trafficking, and cytoskeletal organization. Research into CCDC149 is ongoing, with studies focusing on understanding its cellular localization, binding partners, and potential roles in normal cellular function and disease states. Protein interaction partner analysis indicates that CCDC149 may interact with APP (amyloid precursor protein), PRNP (prion protein), and UBC (ubiquitin C), suggesting potential roles in protein degradation pathways or neurodegenerative processes .

What types of CCDC149 antibodies are available for research?

Currently available CCDC149 antibodies are predominantly rabbit polyclonal antibodies that target various epitopes of the human CCDC149 protein . These include antibodies targeting:

• C-terminal regions (such as ABIN2791254)

• N-terminal regions

• Specific amino acid sequences (e.g., AA 62-89, AA 370-419)

• Internal sequences (e.g., RHQSLKKKYRELIDGDPSLPPEKRKQANLAQLLRDSQDRNKHLGEEIKELQQRLGEVQGDNKLLRMTIAKQRLGDEAIGVRHFAAHER)

Most commercially available antibodies are unconjugated and validated for techniques such as Western blotting, immunohistochemistry, and immunofluorescence .

What species reactivity can be expected from commercial CCDC149 antibodies?

The species reactivity of CCDC149 antibodies varies depending on the specific antibody. Based on the search results, available antibodies show the following reactivity patterns:

Researchers should carefully select antibodies based on their target species and verify cross-reactivity before proceeding with experiments .

What experimental techniques are CCDC149 antibodies validated for?

CCDC149 antibodies have been validated for several common immunological techniques:

These techniques allow researchers to detect and localize CCDC149 protein in cell lysates, tissue sections, and cultured cells. The optimal concentration may vary depending on the specific sample type and experimental conditions, so preliminary titration experiments are recommended .

How should CCDC149 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of CCDC149 antibodies is critical for maintaining their specificity and sensitivity. Based on the search results, the following guidelines should be followed:

• Short-term storage (up to 1 week): Store at 2-8°C

• Long-term storage: Store at -20°C

• Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Some antibodies are supplied in specific buffer conditions:

  • PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose

  • PBS with 40% glycerol and 0.02% sodium azide; pH 7.2

  • Buffered aqueous glycerol solution

• Handle with appropriate precautions, particularly for formulations containing sodium azide, which is a hazardous substance that should be handled by trained personnel only

Proper aliquoting upon receipt can minimize freeze-thaw cycles and extend antibody shelf-life .

What controls should be included when using CCDC149 antibodies in experiments?

Including appropriate controls is essential for interpreting results with CCDC149 antibodies:

• Positive controls:

  • Cell lines or tissues known to express CCDC149

  • Recombinant CCDC149 protein standards

• Negative controls:

  • Samples from knockout models (if available)

  • Isotype controls using non-specific IgG from the same species as the primary antibody

  • Primary antibody omission controls

• Antibody validation controls:

  • Peptide competition assays using the immunizing peptide

  • Comparison of results with multiple antibodies targeting different epitopes

Prestige Antibodies (such as HPA044158) have been extensively validated through the Human Protein Atlas project, which provides tissue array data across 44 normal human tissues and 20 common cancer types, as well as protein array testing against 364 human recombinant protein fragments .

How can specificity issues with CCDC149 antibodies be addressed in challenging experimental contexts?

Antibody specificity is crucial for accurate interpretation of experimental results. For CCDC149 antibodies, researchers can employ these advanced approaches to address specificity concerns:

• Multi-epitope targeting: Use multiple antibodies targeting different regions of CCDC149 (N-terminal, C-terminal, and internal sequences) and compare results .

• Molecular weight verification: CCDC149 has a molecular weight of approximately 58 kDa . Verify that detected bands match this expected size.

• RNA interference validation: Perform siRNA or shRNA knockdown of CCDC149 and confirm reduced signal with the antibody.

• Mass spectrometry validation: For complex samples, immunoprecipitate with the CCDC149 antibody and perform mass spectrometry to confirm target identity.

• Cross-adsorption: For polyclonal antibodies showing cross-reactivity, consider pre-adsorbing against known cross-reactive proteins.

• Signal verification using orthogonal methods: Combine antibody-based detection with non-antibody methods (e.g., RNA-seq or qPCR) to confirm expression patterns.

What is known about CCDC149 expression patterns and how should antibody dilution be optimized accordingly?

CCDC149 expression varies across tissues and cell types, requiring careful optimization of antibody dilutions:

• Expression pattern knowledge: The Human Protein Atlas (referenced for HPA044158) provides comprehensive tissue-specific expression data across 44 normal human tissues and 20 cancer types . Researchers should consult this resource when planning experiments.

• Antibody titration strategy:

  • For tissues with high CCDC149 expression: Begin with higher dilutions (1:500 for IHC, 0.04 μg/mL for WB)

  • For tissues with low expression: Use more concentrated antibody (1:200 for IHC, 0.4 μg/mL for WB)

  • Always perform a dilution series (typically 2-fold or 3-fold) to determine optimal signal-to-noise ratio

• Sample-specific considerations:

  • Fresh frozen vs. FFPE tissues may require different antibody concentrations

  • Different fixation methods may affect epitope accessibility

  • Cell lines vs. primary cells may show different expression levels

• Signal amplification: For tissues with very low expression, consider using signal amplification methods (e.g., tyramide signal amplification) rather than simply increasing antibody concentration, which may increase background.

How can CCDC149 antibodies be integrated into multiplexing approaches for studying protein interactions?

Advanced multiplexing techniques can provide insights into CCDC149 interactions with other proteins:

• Co-immunoprecipitation strategies: CCDC149 has reported interactions with APP, PRNP, and UBC . Co-IP using CCDC149 antibodies followed by Western blotting for these potential interaction partners can validate these interactions.

• Multiplexed immunofluorescence:

  • Select CCDC149 antibodies raised in different host species than antibodies against potential interaction partners

  • Use directly conjugated secondary antibodies with minimal cross-reactivity

  • Consider sequential staining protocols for challenging combinations

  • Use spectral unmixing for fluorophores with overlapping emission spectra

• Proximity ligation assay (PLA):

  • Ideal for detecting protein-protein interactions in situ

  • Requires antibodies against CCDC149 and interaction partner from different species

  • Generates fluorescent signal only when proteins are within 40 nm

• CODEX or Imaging Mass Cytometry:

  • For highly multiplexed approaches (>10 proteins)

  • Requires optimization of CCDC149 antibody staining within panel

  • Consider epitope retrieval compatibility with other target proteins

What are common pitfalls when using CCDC149 antibodies in Western blotting and how can they be resolved?

Western blotting with CCDC149 antibodies may encounter several challenges:

• Multiple bands or unexpected molecular weight:

  • CCDC149 has a predicted molecular weight of 58 kDa

  • Unexpected bands could represent post-translational modifications, splice variants, degradation products, or non-specific binding

  • Verify specificity using knockout/knockdown controls or peptide competition assays

  • Optimize sample preparation to minimize protein degradation (use fresh protease inhibitors)

• Weak or no signal:

  • Increase antibody concentration within recommended range (0.04-0.4 μg/mL)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure protein transfer efficiency with reversible staining

  • Consider different epitope antibodies if particular epitope might be masked

  • Optimize protein extraction method for membrane proteins with coiled-coil domains

• High background:

  • Increase blocking time or concentration (5% non-fat milk or BSA)

  • Increase wash duration and number of washes

  • Reduce primary and secondary antibody concentrations

  • Consider using different blocking reagents (casein, commercial blockers)

  • Filter antibody solutions before use to remove aggregates

• Sample-specific issues:

  • For tissues with high lipid content, optimize extraction protocols

  • For samples with high proteolytic activity, adjust protease inhibitor cocktail

How can epitope masking issues be addressed in immunohistochemistry with CCDC149 antibodies?

Epitope masking is a common challenge in immunohistochemistry that can affect CCDC149 detection:

• Antigen retrieval optimization:

  • Test multiple retrieval methods: heat-induced (citrate buffer pH 6.0, EDTA buffer pH 9.0) and enzymatic (proteinase K, trypsin)

  • Optimize retrieval time and temperature (e.g., 10-30 minutes at 95-100°C)

  • For CCDC149, which has coiled-coil domains that may be sensitive to fixation, EDTA-based retrieval at pH 9.0 may be more effective

• Fixation considerations:

  • Excessive formalin fixation can cause cross-linking that masks epitopes

  • Consider testing samples with different fixation durations

  • For prospective studies, optimize fixation protocol (4% paraformaldehyde for 24-48 hours)

• Antibody penetration:

  • For thick sections, increase incubation time or consider antigen retrieval with detergents

  • Test different detergents in wash buffers (0.1-0.3% Triton X-100, 0.05-0.1% Tween-20)

• Sequential epitope retrieval:

  • For challenging epitopes, consider performing multiple rounds of antigen retrieval

  • Allow cooling between cycles to prevent tissue detachment

What strategies can address non-specific binding issues with CCDC149 polyclonal antibodies?

Polyclonal antibodies against CCDC149 may exhibit non-specific binding that requires advanced troubleshooting:

• Pre-adsorption techniques:

  • Incubate antibody with tissues or cell lysates from species with low homology to human CCDC149

  • Pre-adsorb against common cross-reactive epitopes (particularly important for antibodies targeting coiled-coil domains, which share structural similarities)

• Blocking optimization:

  • Test species-specific serum matching the host species of secondary antibody

  • Include protein from the same species as the primary antibody in blocking solution

  • Use commercial blocking solutions designed to reduce non-specific binding

• Antibody purification:

  • Consider using affinity-purified antibodies (like ABIN2791254, which is affinity purified)

  • Protein A/G purification may help reduce non-specific binding

• Extraction method considerations:

  • Optimize lysis buffers to reduce co-extraction of cross-reactive proteins

  • Consider membrane fractionation methods for membrane-associated proteins

• Signal verification:

  • Compare staining patterns between multiple antibodies against different CCDC149 epitopes

  • Consider monoclonal antibody alternatives if available

How can CCDC149 antibodies be effectively used in studying protein-protein interactions?

Investigating CCDC149 interactions with reported partners (APP, PRNP, UBC) requires specialized approaches:

• Co-immunoprecipitation optimization:

  • Select antibodies with minimal interference with protein interaction sites

  • Test both N-terminal and C-terminal targeting antibodies

  • Optimize lysis conditions to preserve protein complexes (mild detergents like 0.5% NP-40)

  • Consider cross-linking approaches for transient interactions

  • Validate results bidirectionally (IP with CCDC149 antibody and blot for partner, then IP with partner antibody and blot for CCDC149)

• Chromatin immunoprecipitation (ChIP) adaptations:

  • For potential nuclear functions of CCDC149

  • Optimize sonication conditions for coiled-coil domain preservation

  • Verify antibody compatibility with crosslinking reagents

• FRET-based interaction studies:

  • Select antibodies compatible with direct fluorophore conjugation

  • Optimize antibody-fluorophore ratio to maintain binding while enabling energy transfer

  • Control for potential steric hindrance effects

• Label-free interaction analysis:

  • Surface Plasmon Resonance (SPR) using purified CCDC149 and immobilized antibodies

  • Biolayer Interferometry with biotinylated antibodies

What considerations are important when designing knockdown/knockout validation experiments for CCDC149 antibodies?

Genetic manipulation approaches provide powerful validation tools for CCDC149 antibodies:

• siRNA/shRNA knockdown design:

  • Target conserved exons present in all known CCDC149 splice variants

  • Design multiple independent siRNAs and validate knockdown efficiency by qPCR

  • Optimize transfection conditions for cell types of interest

  • Include scrambled siRNA controls

• CRISPR/Cas9 knockout considerations:

  • Design guide RNAs targeting early exons to ensure complete protein disruption

  • Create epitope-specific knockouts by targeting regions recognized by specific antibodies

  • Generate homozygous and heterozygous knockout lines to create signal gradient

  • Verify knockout by sequencing and transcript analysis before antibody validation

• Validation experimental design:

  • Compare antibody signal in wild-type vs. knockdown/knockout samples across multiple techniques

  • Quantify signal reduction and correlate with mRNA reduction levels

  • Consider time course experiments to account for protein stability

• Potential challenges:

  • Essential gene functions may prevent complete knockout

  • Compensatory upregulation of related proteins

  • Off-target effects of genetic manipulation tools

How might CCDC149 antibodies be adapted for advanced imaging techniques in research?

Emerging imaging technologies offer new possibilities for CCDC149 localization studies:

• Super-resolution microscopy adaptations:

  • STORM/PALM: Use antibodies conjugated with photoswitchable fluorophores

  • STED: Select antibodies compatible with high-power depletion lasers

  • SIM: Optimize antibody concentration for high signal-to-noise ratio

  • Consider direct labeling strategies to reduce spatial displacement due to primary-secondary antibody combinations

• Live-cell imaging approaches:

  • Develop cell-permeable antibody fragments (Fab, nanobodies)

  • Optimize antibody loading techniques (microinjection, electroporation)

  • Consider epitope-tagging strategies combined with fluorescent protein fusions

• Correlative light and electron microscopy (CLEM):

  • Select antibodies compatible with EM fixation and embedding protocols

  • Test performance with gold-conjugated secondary antibodies

  • Optimize permeabilization conditions for intracellular epitopes

• Expansion microscopy:

  • Verify antibody compatibility with hydrogel embedding

  • Test epitope preservation after expansion

  • Adjust antibody concentration for expanded samples