C2orf49 Antibody

What is C2orf49 protein and why is it relevant for research?

C2orf49, also known as Ashwin, is a 232 amino acid protein encoded by a gene located on human chromosome 2q12.1. It belongs to the ashwin family and is associated with nucleoplasmic and tRNA-splicing ligase complex components . The protein has a calculated molecular weight of 26 kDa, though it typically appears between 25-30 kDa in experimental conditions . Despite being relatively understudied, C2orf49 has potential roles in cell signaling, metabolism, tRNA processing, and gene expression pathways, making it an important target for research into fundamental cellular processes .

What experimental applications are C2orf49 antibodies suitable for?

C2orf49 antibodies have been validated for multiple experimental applications. Western Blot (WB) applications typically use dilutions ranging from 1:1000 to 1:8000, with positive detection in human cell lines like HeLa and Jurkat, as well as mouse liver tissue . Immunofluorescence (IF) and immunocytochemistry (ICC) applications utilize dilutions between 1:300 and 1:1200, with successful detection in A431 cells . Additionally, some C2orf49 antibodies have been validated for ELISA (dilutions 1:2000-1:10000) and immunohistochemistry (IHC) applications (dilutions 1:20-1:200) . It is important to optimize antibody concentrations for each specific experimental system to achieve optimal results .

What are the species reactivity profiles of commercially available C2orf49 antibodies?

Most commercially available C2orf49 antibodies show reactivity with human, mouse, and rat samples . The sequence homology between human C2orf49 and its orthologs is relatively high, with mouse sharing 76% identity and rat sharing 78% identity in the antigen sequence regions . This cross-reactivity makes these antibodies valuable for comparative studies across species, though researchers should validate antibody performance in their specific experimental model .

What are the optimal sample preparation procedures for C2orf49 detection in Western blotting?

For optimal detection of C2orf49 in Western blotting, prepare protein lysates under denaturing conditions using standard SDS-PAGE protocols. Various lysates (HeLa cells, mouse liver tissue, Jurkat cells) have been successfully used for C2orf49 detection . A recommended protocol involves subjecting lysates to SDS-PAGE followed by Western blot with the antibody at dilution of 1:4000, incubated at room temperature for 1.5 hours . When analyzing results, expect to observe the C2orf49 protein at approximately 25-30 kDa on your blot, which aligns with the observed molecular weight reported in validation studies .

How should immunofluorescence experiments be optimized for C2orf49 detection?

For immunofluorescence detection of C2orf49, 4% paraformaldehyde (PFA) fixation has been successfully used with cells such as A431 . A validated protocol uses C2orf49 antibody at a dilution of 1:600 along with appropriate fluorophore-conjugated secondary antibodies (e.g., CoraLite®488-Conjugated Goat Anti-Rabbit IgG) . When designing multiplex staining experiments, C2orf49 antibody has been successfully combined with cytoskeletal markers like phalloidin (CL594-Phalloidin) . Researchers should include appropriate controls and optimize fixation conditions based on their specific cell type and experimental requirements.

What are the recommended storage conditions to maintain antibody stability and performance?

To maintain optimal stability and performance of C2orf49 antibodies, store at -20°C in the provided storage buffer, which typically contains PBS with glycerol (50%) and small amounts of sodium azide (0.02-0.1%) . Most manufacturers indicate that the antibodies are stable for one year after shipment when properly stored . While aliquoting is specified as unnecessary for -20°C storage in some cases, it may still be beneficial to minimize freeze-thaw cycles . Some antibody preparations contain 0.1% BSA as a stabilizer in smaller volumes (20μl sizes) . Always follow manufacturer-specific storage recommendations as buffer compositions may vary slightly between suppliers.

How can researchers address non-specific binding when using C2orf49 antibodies?

When encountering non-specific binding with C2orf49 antibodies, implement a comprehensive optimization strategy. First, titrate the antibody concentration by testing a broader dilution range than the recommended 1:1000-1:8000 for WB or 1:300-1:1200 for IF/ICC . Second, optimize blocking conditions by testing different blocking agents (BSA, milk, serum) and increasing blocking time. Third, ensure adequate washing steps between antibody incubations using PBS-T or TBS-T buffers. Additionally, consider the purification method of your antibody - C2orf49 antibodies generated through antigen affinity purification methods may provide higher specificity . Lastly, include proper controls in your experimental design, such as knockdown/knockout samples or peptide competition assays.

What strategies can improve detection sensitivity for low-abundance C2orf49 expression?

For improved detection of low-abundance C2orf49, employ multiple technical enhancements. First, consider signal amplification methods such as using high-sensitivity ECL substrates for Western blot or tyramide signal amplification for IHC/IF applications. Second, optimize protein extraction methods to ensure efficient recovery of nuclear proteins, as C2orf49 has been associated with the nucleoplasm . Third, enrich your sample through immunoprecipitation before Western blot analysis. Fourth, increase antibody incubation time and temperature parameters to enhance binding kinetics. Finally, consider using more sensitive detection methods like automated Western blot systems or digital immunoassay platforms that provide greater sensitivity than traditional methods.

How can researchers validate antibody specificity for C2orf49?

To validate C2orf49 antibody specificity, implement a multi-faceted validation approach. First, perform Western blot analysis looking for a single band at the expected molecular weight (25-30 kDa) . Second, conduct peptide competition assays using the immunogen sequence from which the antibody was derived. Antibodies like PA5-66348 have published immunogen sequences (e.g., "GSSTSTSIKVK KTENGDNDRL KPPPQASFTS NAFRKLSNSS SSVSPLILSS NLPVNNKTEH NNNDAKQNHD LTHRKSPSGP VKSPPLSPVG T") that can be synthesized for blocking experiments . Third, use genetic approaches by testing antibody reactivity in C2orf49 knockdown/knockout cells compared to wild-type controls. Fourth, compare staining patterns across multiple C2orf49 antibodies targeting different epitopes. Finally, verify subcellular localization patterns align with expected nucleoplasmic and tRNA-splicing ligase complex locations .

How can C2orf49 antibodies be utilized to investigate potential roles in tRNA processing?

To investigate C2orf49's role in tRNA processing, implement a multi-faceted experimental approach. First, conduct co-immunoprecipitation studies using C2orf49 antibodies to identify interacting partners within the tRNA-splicing ligase complex . Second, perform chromatin immunoprecipitation (ChIP) assays to examine C2orf49 association with relevant genomic regions involved in tRNA expression. Third, use cellular fractionation followed by immunoblotting with C2orf49 antibodies (1:1000-1:4000 dilution) to confirm its presence in nucleoplasm and tRNA-processing compartments . Fourth, establish cell models with C2orf49 knockout/knockdown and analyze tRNA processing efficiency using northern blotting or RNA-seq approaches. Finally, conduct immunofluorescence co-localization studies using C2orf49 antibodies (1:300-1:600 dilution) alongside markers for tRNA processing bodies to visualize spatial relationships .

What experimental designs can explore potential roles of C2orf49 in disease pathways?

To explore C2orf49's potential involvement in disease pathways, implement a comprehensive research strategy. First, analyze C2orf49 expression levels across disease-relevant tissues using immunohistochemistry (IHC) with validated antibodies at 1:20-1:200 dilutions . Second, conduct tissue microarray (TMA) analysis to compare C2orf49 expression patterns across multiple disease states and normal tissues. Third, perform correlation studies between C2orf49 expression levels (determined by Western blot) and disease progression markers. Fourth, establish disease-relevant cell models (e.g., cancer cell lines, primary patient cells) and manipulate C2orf49 expression to observe phenotypic changes. Fifth, use phospho-specific antibodies or conduct post-translational modification analysis to determine if C2orf49 undergoes regulatory modifications during disease states. Finally, perform high-throughput screens to identify compounds that modulate C2orf49 function, potentially revealing therapeutic targets.

How can researchers investigate potential C2orf49 post-translational modifications?

To investigate post-translational modifications (PTMs) of C2orf49, implement a systematic analytical approach. First, perform immunoprecipitation using C2orf49 antibodies followed by mass spectrometry analysis to identify potential modification sites. Second, conduct Western blot analysis under conditions that preserve PTMs (e.g., including phosphatase inhibitors, deacetylase inhibitors) using the standard dilution range of 1:1000-1:8000 . Third, use specific PTM detection methods such as Phos-tag gels for phosphorylation analysis or ubiquitin-specific antibodies for ubiquitination studies. Fourth, generate or obtain antibodies specific to predicted PTM sites on C2orf49. Fifth, perform in vitro enzymatic assays with purified C2orf49 and candidate modifying enzymes to confirm direct modification. Finally, analyze the functional consequences of these modifications through site-directed mutagenesis of key residues followed by functional assays relevant to C2orf49's proposed roles in tRNA processing and gene expression .

What are the key technical specifications to consider when selecting a C2orf49 antibody?

When selecting a C2orf49 antibody, consider several critical technical specifications. First, evaluate the immunogen used to generate the antibody - some are raised against full-length fusion proteins (e.g., AG12948) , while others target specific peptide sequences (e.g., "GSSTSTSIKVK KTENGDNDRL KPPPQASFTS NAFRKLSNSS SSVSPLILSS NLPVNNKTEH NNNDAKQNHD LTHRKSPSGP VKSPPLSPVG T") . Second, assess clonality - most available C2orf49 antibodies are polyclonal, raised in rabbits, providing broad epitope recognition . Third, verify the purification method, with antigen affinity purification typically providing higher specificity . Fourth, examine species reactivity, with most antibodies showing cross-reactivity with human, mouse, and rat samples . Fifth, confirm validated applications (WB, IF/ICC, ELISA, IHC) with their appropriate dilution ranges . Finally, check RRID (Research Resource Identifiers) such as AB_2066525 to ensure you can properly cite and track the specific antibody reagent in your research.

What buffer systems and reagents are compatible with C2orf49 antibodies?

C2orf49 antibodies are compatible with standard buffer systems used in immunological techniques. For Western blotting, standard SDS-PAGE and transfer buffers work effectively with recommended antibody dilutions of 1:1000-1:8000 . For immunofluorescence, PBS-based buffers with 4% PFA fixation have been validated . Most C2orf49 antibodies are stored in PBS with 0.02-0.1% sodium azide and 50% glycerol at pH 7.3 , making them compatible with most standard immunoassay diluents. When selecting blocking reagents, both BSA and non-fat dry milk have been used successfully, though specific optimization may be required for your experimental system . For detection systems, both chemiluminescence (for WB) and fluorophore-conjugated secondary antibodies (for IF/ICC) have demonstrated compatibility with C2orf49 primary antibodies .

What experimental factors might affect the performance of C2orf49 antibodies?

Multiple experimental factors can significantly impact C2orf49 antibody performance. First, fixation methods - while 4% PFA has been validated for immunofluorescence , alternative fixatives may affect epitope accessibility. Second, protein extraction methods - as C2orf49 is associated with nucleoplasm and tRNA-splicing complexes , extraction protocols optimized for nuclear proteins may improve detection. Third, sample processing - excessive freeze-thaw cycles of tissue/cell lysates may degrade the target protein. Fourth, incubation parameters - temperature, time, and agitation conditions should be optimized for each application. Fifth, secondary antibody selection - ensure proper host species matching and minimal cross-reactivity. Sixth, blocking conditions - insufficient blocking may increase background. Finally, reagent quality - degradation of antibodies through improper storage or handling can significantly reduce performance. Researchers should systematically optimize these factors for their specific experimental systems to achieve reproducible results.