SPAC7D4.08 Antibody

What is SPAC7D4.08 antibody and what organism does it target?

SPAC7D4.08 antibody is a polyclonal antibody that specifically targets the SPAC7D4.08 protein from Schizosaccharomyces pombe (fission yeast). The antibody is developed using a recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPAC7D4.08 protein as the immunogen. It is produced in rabbit hosts and purified using antigen affinity chromatography, resulting in high specificity for the target protein. This antibody recognizes epitopes of the SPAC7D4.08 protein, which is encoded by the gene with Entrez Gene ID 2542739 and UniProt number O14263. The polyclonal nature of this antibody means it recognizes multiple epitopes on the target protein, which can provide robust detection even if some epitopes are altered or obscured .

What applications is the SPAC7D4.08 antibody validated for?

The SPAC7D4.08 antibody has been validated for specific applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB). These techniques allow researchers to detect and quantify the SPAC7D4.08 protein in various experimental contexts. In ELISA applications, the antibody can be used to detect the target protein in solution, providing quantitative or semi-quantitative data depending on the specific ELISA format employed. For Western blotting, the antibody enables detection of the denatured protein following electrophoretic separation, providing information about molecular weight and relative abundance. Although not explicitly validated for other applications like immunohistochemistry or immunofluorescence, researchers might explore these applications with appropriate validation controls .

What components are included in the SPAC7D4.08 antibody package?

The SPAC7D4.08 antibody package typically contains three essential components:

• 200μg antigens (used as positive control): This component allows researchers to validate the antibody's specificity and optimize experimental conditions.

• 1ml pre-immune serum (used as negative control): This serum, collected from the host animal before immunization, serves as a crucial negative control to distinguish between specific and non-specific binding.

• Rabbit polyclonal antibodies purified by Antigen Affinity: The primary antibody that has been purified using affinity chromatography to enhance specificity.

These components collectively enable researchers to implement proper experimental controls, which are essential for result validation and troubleshooting. The inclusion of both positive and negative controls represents best practices in antibody-based research approaches .

How should I design ELISA experiments using SPAC7D4.08 antibody?

When designing ELISA experiments with SPAC7D4.08 antibody, follow a systematic approach to ensure reliable results. Begin with a standard indirect ELISA by coating plates with your antigen (protein lysate containing SPAC7D4.08) or use a sandwich ELISA approach if detecting the protein in solution. Establish appropriate blocking conditions (typically 1-5% BSA or non-fat milk) to minimize non-specific binding. Create a dilution series of the SPAC7D4.08 antibody (starting from 1:100 to 1:10,000) to determine optimal concentration for specific signal with minimal background. Include both positive and negative controls—use the provided 200μg antigen as positive control and pre-immune serum as negative control. Develop a standard curve using purified recombinant protein if quantitative results are needed. For detection, select secondary antibodies suitable for rabbit IgG, such as goat anti-rabbit IgG conjugated with alkaline phosphatase or HRP .

For sandwich ELISA formats, consider pairing with another antibody recognizing a different epitope, similar to approaches used for other proteins in the literature . Document all experimental parameters including coating buffers, blocking buffers, antibody dilutions, incubation times and temperatures, and washing procedures to ensure reproducibility .

What is the recommended protocol for Western blotting with SPAC7D4.08 antibody?

For Western blotting with SPAC7D4.08 antibody, begin sample preparation by extracting proteins under conditions that preserve the target protein's integrity. Separate proteins using SDS-PAGE (10-12% gel recommended for most standard-sized proteins) and transfer to a PVDF or nitrocellulose membrane. Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Dilute the SPAC7D4.08 antibody to an appropriate concentration (start with 1:500 to 1:2000) in blocking buffer and incubate overnight at 4°C with gentle agitation. Wash the membrane 3-5 times with TBST, then incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000 to 1:10000) for 1 hour at room temperature .

Include the provided positive control antigen in one lane to confirm antibody specificity. After washing, develop using ECL substrate and image using film or digital imaging system. If working with crude cell lysates, include appropriate controls such as knockout/knockdown samples or pre-immune serum to distinguish specific from non-specific bands. Optimize antibody concentration, incubation time, and washing steps based on initial results. For quantitative analysis, consider using loading controls appropriate for your experimental system .

How should I validate the specificity of SPAC7D4.08 antibody for my particular research application?

Validating SPAC7D4.08 antibody specificity requires multiple approaches to ensure robust and reproducible results. Begin with a literature search to identify previously validated methods and reported molecular weights for your target protein. Perform Western blot analysis using the provided positive control antigen alongside your experimental samples to confirm recognition of the expected molecular weight band. Include negative controls such as lysates from cells or organisms known not to express the target protein, or lysates where the target has been knocked down/out via genetic methods .

For more rigorous validation, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody captures the intended target. Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before use in your application, can further confirm specificity (signal should be abolished or significantly reduced). For applications beyond Western blot and ELISA, such as immunofluorescence, validate localization patterns against known literature or with orthogonal methods. Document all validation steps methodically and include these controls in your experimental design to enhance reproducibility and confidence in your results .

What are the optimal storage conditions for SPAC7D4.08 antibody?

SPAC7D4.08 antibody requires careful storage conditions to maintain its functionality and specificity over time. The recommended storage temperature is -20°C or -80°C, with -80°C being preferable for long-term storage to prevent degradation. When storing at -20°C, minimize freeze-thaw cycles by aliquoting the antibody into smaller volumes upon receipt. Each freeze-thaw cycle can potentially diminish antibody activity through protein denaturation and aggregation. For working solutions, short-term storage at 4°C (up to one week) is acceptable, but longer periods should be avoided. The antibody should be stored in its original buffer formulation, which typically contains stabilizers and preservatives to maintain antibody integrity. When handling the antibody, allow it to equilibrate to room temperature before opening the vial to prevent condensation, which can introduce contaminants and accelerate degradation .

How should I prepare working dilutions of SPAC7D4.08 antibody?

Preparation of working dilutions of SPAC7D4.08 antibody requires careful attention to maintain antibody performance. Begin by allowing the stock antibody to thaw completely at 4°C (avoid room temperature thawing which can promote degradation). Gently mix the antibody by inversion or mild vortexing to ensure homogeneity, but avoid vigorous shaking that could cause protein denaturation or foaming. Prepare working dilutions in clean tubes using high-quality buffers—typically PBS or TBS containing 0.1-5% BSA as a carrier protein to prevent antibody adsorption to tube walls. For ELISA applications, start with dilutions ranging from 1:500 to 1:5000, while Western blotting typically requires 1:500 to 1:2000 dilutions .

Prepare fresh working dilutions for each experiment rather than storing diluted antibody for extended periods. If storage of diluted antibody is necessary, add stabilizing proteins (BSA) and preservatives (sodium azide at 0.02-0.05%, being mindful that azide can inhibit HRP activity). Document the exact dilution protocol, buffer composition, and handling conditions to ensure experimental reproducibility across multiple sessions .

How can I address high background issues when using SPAC7D4.08 antibody in immunoassays?

High background is a common challenge when working with antibodies like SPAC7D4.08. To address this issue systematically, first evaluate the blocking step by testing different blocking agents (BSA, non-fat milk, casein, or commercial blockers) at various concentrations (1-5%). Increase the blocking time from the standard 1 hour to 2-3 hours at room temperature or overnight at 4°C. Next, optimize antibody dilutions—use a higher dilution of the primary antibody (start by doubling your current dilution) and ensure secondary antibody is appropriately diluted (typically 1:5000-1:10000). Increase the number and duration of washing steps using buffers containing slightly higher detergent concentrations (0.1-0.5% Tween-20 in PBS/TBS) .

Test the pre-immune serum provided as a negative control at the same concentration as your primary antibody to determine if the background is antibody-specific. If using colorimetric detection systems, reduce substrate incubation time or concentration. For Western blots specifically, pre-adsorb the antibody with membrane proteins from a non-target species or use highly purified samples. Document successful modifications to your protocol to establish reproducible conditions for future experiments .

What strategies should I employ if I'm detecting weak or no signal with SPAC7D4.08 antibody?

When encountering weak or no signal with SPAC7D4.08 antibody, implement a systematic troubleshooting approach. First, confirm antibody viability by testing the provided positive control antigen in a standard protocol. Verify protein expression and loading—ensure sufficient target protein is present by staining gels with Coomassie blue before transfer, or use alternative detection methods to confirm protein expression. Optimize antigen retrieval for your sample type; for yeast proteins, consider different lysis methods that effectively solubilize membrane or structural proteins .

Decrease antibody dilution incrementally (try 1:250 if 1:500 shows no signal) and extend primary antibody incubation time (overnight at 4°C instead of 1-2 hours). Enhance detection sensitivity by using amplification systems such as biotin-streptavidin, or switch to more sensitive detection reagents like ECL-Plus for Western blots. For ELISA, consider switching from colorimetric to chemiluminescent detection. Optimize buffer conditions—test different pH values and salt concentrations that might better preserve epitope structure. Verify that your secondary antibody recognizes rabbit IgG properly and is functional using positive controls .

If issues persist, consider epitope accessibility problems—milder denaturation conditions for Western blots or alternative fixation methods for immunohistochemistry might preserve epitope structure better. As a last resort, test alternative lots of the antibody or contact the manufacturer for technical support .

How can I use SPAC7D4.08 antibody in multiplexed immunoassays?

Implementing SPAC7D4.08 antibody in multiplexed immunoassays requires careful planning to avoid cross-reactivity and ensure signal specificity. Begin by conjugating the antibody with a distinct fluorophore, biotin, or another distinguishable tag if direct detection is preferred. Alternatively, use isotype-specific secondary antibodies when combining with other primary antibodies of different host species. For multiplex flow cytometry, use fluorophores with minimal spectral overlap and include proper compensation controls. In multiplex Western blotting, consider size differences between target proteins and use different color detection systems for each target .

When designing multiplex ELISA, use the sandwich format with capturing antibodies of different specificities on encoded beads or defined spatial locations. Validate each antibody individually before combining them, and always run single-target controls alongside multiplexed experiments to identify any interference effects. Consider cross-adsorption of secondary antibodies against other species' immunoglobulins to minimize cross-reactivity when multiple primary antibodies are used simultaneously. Thorough washing between primary and secondary antibody application is critical in multiplexed systems to reduce background and cross-reactivity. Document optimization steps and validation methods specifically for the multiplex context, as conditions may differ from single-target applications .

What approaches are recommended for quantitative analysis using SPAC7D4.08 antibody?

For quantitative analysis using SPAC7D4.08 antibody, implement a multi-faceted approach to ensure accuracy and reproducibility. For ELISA-based quantification, develop a standard curve using purified recombinant SPAC7D4.08 protein at known concentrations (typically ranging from 0.1 ng/mL to 1000 ng/mL). Use four-parameter logistic regression for curve fitting rather than simple linear regression to account for the sigmoidal relationship between concentration and signal. Run all samples in triplicate and include quality control samples of known concentration to monitor assay performance across multiple runs .

For Western blot quantification, use internal loading controls appropriate for your experimental context (housekeeping proteins like GAPDH for total protein normalization, or spiked-in control proteins for absolute quantification). Employ digital imaging systems with a broad dynamic range rather than film-based detection, and validate that signal response is linear across your expected concentration range. Perform densitometry analysis using software that can distinguish specific signal from background, and normalize target protein measurements to loading controls .

For more advanced applications like flow cytometry, use antibody binding capacity (ABC) beads to establish a calibration curve that converts fluorescence intensity to absolute number of bound antibodies. Regardless of the application, include biological replicates and appropriate statistical analysis to account for natural variation. Report detailed methods, including quantification approaches, software used, and statistical analyses performed, to enable others to reproduce your quantitative results .

How can I optimize SPAC7D4.08 antibody for detecting low-abundance targets?

Detecting low-abundance targets with SPAC7D4.08 antibody requires specialized approaches to enhance sensitivity while maintaining specificity. Begin by enriching your target protein through subcellular fractionation, immunoprecipitation, or affinity purification techniques before analysis. For Western blotting, increase protein loading (up to 50-100 μg per lane) and use high-sensitivity ECL substrates or fluorescent detection systems with digital imaging. Consider using gradient gels to improve separation and concentration of target proteins within a narrower band .

For ELISA applications, implement signal amplification strategies such as biotin-streptavidin systems, tyramide signal amplification, or poly-HRP conjugates that can significantly increase detection sensitivity. Extend primary antibody incubation time to overnight at 4°C to allow more complete binding to rare targets, and optimize blocking and washing conditions to minimize background while preserving specific signals. Using a sandwich ELISA format with a capture antibody can also concentrate the target from dilute samples .

Additionally, consider digital PCR or proximity ligation assays as complementary approaches for validating low-abundance protein detection. Throughout optimization, maintain rigorous controls including dilution series of positive controls to define the lower limit of detection, and negative controls to ensure signal specificity. Document detailed protocols including all amplification steps, incubation conditions, and detection parameters to ensure reproducibility of your enhanced sensitivity methods .

How does SPAC7D4.08 polyclonal antibody compare with monoclonal alternatives for research applications?

Monoclonal antibodies, while offering higher specificity and consistency between batches, recognize only a single epitope, which may be masked or altered under certain experimental conditions. For quantitative applications requiring precise reproducibility over time, monoclonal antibodies generally provide more consistent results. For applications like sandwich ELISA, using a combination of polyclonal capture antibody with monoclonal detection antibody can leverage the advantages of both types .

When selecting between polyclonal and monoclonal options, consider your experimental requirements—use polyclonal antibodies like SPAC7D4.08 when sensitivity and robust detection are priorities, particularly in applications involving denatured proteins. Choose monoclonal alternatives when absolute specificity and reproducibility are paramount, especially for long-term studies requiring consistent reagents .

What orthogonal methods can complement SPAC7D4.08 antibody-based detection for validation?

Recombinant expression systems using tagged versions of the target protein (e.g., GFP-tagged or epitope-tagged SPAC7D4.08) allow parallel detection with anti-tag antibodies to confirm localization or expression patterns. Proximity ligation assays can validate protein-protein interactions initially detected through co-immunoprecipitation with SPAC7D4.08 antibody. For functional validation, complement immunodetection with activity assays specific to your protein's known biochemical function .

What minimum validation data should researchers report when publishing results using SPAC7D4.08 antibody?

When publishing results using SPAC7D4.08 antibody, researchers should report comprehensive validation data to enhance reproducibility and scientific rigor. At minimum, include the complete antibody identifier information including supplier (Cusabio), catalog number (CSB-PA519379XA01SXV-2), lot number, and RRID if available. Document the validation experiments performed specifically for your application, including Western blot images showing the detection of bands at expected molecular weights, with positive and negative controls clearly labeled. For ELISA applications, report standard curves demonstrating linear range, limit of detection, and specificity tests using competition assays or knockout samples .

Include detailed methods sections describing antibody dilution (1:500, 1:1000, etc.), incubation conditions (time, temperature), buffer compositions, blocking reagents, and detection systems used. Report any optimization steps undertaken to enhance specificity or sensitivity. For applications beyond the manufacturer's validated uses, provide additional validation data demonstrating the antibody's suitability for these purposes. If using the antibody for quantitative analysis, document the reproducibility between technical and biological replicates, including appropriate statistical analyses .

This comprehensive reporting enables other researchers to accurately reproduce your methods and properly interpret your results, addressing a significant challenge in antibody-based research. Following these standards aligns with current best practices in antibody validation and reporting as emphasized in recent scientific literature .

How should researchers address potential cross-reactivity concerns with SPAC7D4.08 antibody?

Addressing cross-reactivity concerns with SPAC7D4.08 antibody requires systematic investigation and transparent reporting. Begin by conducting cross-reactivity tests against proteins with sequence similarity to SPAC7D4.08, particularly if working in systems that express related proteins. Perform Western blot analysis using lysates from multiple species or cell types to identify any unexpected bands that might indicate cross-reactivity. When possible, include genetic controls such as knockout or knockdown samples to confirm that detected signals are specific to SPAC7D4.08 .

For more comprehensive assessment, conduct peptide competition assays where the antibody is pre-incubated with excess immunizing peptide before use; specific signals should be abolished while cross-reactive signals may persist. Consider immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody. For polyclonal antibodies like SPAC7D4.08, affinity purification against the specific antigen can reduce cross-reactivity .

In publications, transparently report all cross-reactivity testing performed and any observed cross-reactivity. Include images of full Western blots (not just the region of interest) to allow readers to evaluate potential cross-reactive bands. Specify the concentrations at which any cross-reactivity was observed and whether it impacts the interpretation of your results. This thorough approach to addressing cross-reactivity concerns enhances research reliability and facilitates proper interpretation of experimental results by the scientific community .

What statistical approaches are recommended for analyzing quantitative data generated using SPAC7D4.08 antibody?

For Western blot densitometry data, normalize target protein measurements to appropriate loading controls before statistical comparison. Account for the limited dynamic range of Western blot by verifying that measurements fall within the linear range of detection. When comparing multiple experimental conditions, apply ANOVA with appropriate post-hoc tests (Tukey's, Bonferroni) to correct for multiple comparisons and control Type I error rates .

For all analyses, report effect sizes alongside p-values to indicate biological significance beyond statistical significance. Include detailed descriptions of statistical methods, software packages used, normalization procedures, and handling of outliers. Consider implementing more advanced approaches like mixed-effects models for experiments with complex designs involving multiple factors or repeated measures. This comprehensive statistical approach enhances the reliability and interpretability of quantitative data generated using SPAC7D4.08 antibody .

How should researchers interpret unexpected results or discrepancies when using SPAC7D4.08 antibody?

Investigate biological explanations for unexpected results—post-translational modifications, alternative splicing, or protein-protein interactions might mask epitopes or alter apparent molecular weights. For context-dependent discrepancies, examine experimental conditions including cell/tissue types, developmental stages, or treatment conditions that might affect protein expression or localization. Implement orthogonal detection methods to determine whether the discrepancy is antibody-specific or reflects true biological variation .

Document all troubleshooting steps methodically and report unexpected findings transparently in publications, as these may represent novel biological insights rather than technical artifacts. Engage with the scientific community and the antibody manufacturer when persistent discrepancies arise, as others may have encountered similar issues. This methodical approach to interpreting unexpected results not only resolves technical issues but may also lead to new biological discoveries .

How might SPAC7D4.08 antibody be adapted for advanced imaging techniques?

Adapting SPAC7D4.08 antibody for advanced imaging techniques requires specialized modification and validation approaches. For super-resolution microscopy (STORM, PALM, or SIM), consider direct conjugation with bright, photostable fluorophores like Alexa Fluor 647 or Atto dyes that provide the necessary photophysical properties for single-molecule localization. Validate labeling density and specificity at the nanoscale resolution these techniques provide, as background and non-specific binding become more apparent. For expansion microscopy, test compatibility with the hydrogel embedding process and protein digestion steps to ensure epitope preservation .

For intravital imaging applications, consider conjugation with near-infrared fluorophores that provide better tissue penetration and lower autofluorescence. For multiplexed imaging such as Imaging Mass Cytometry or CODEX, conjugate the antibody with rare earth metals or DNA barcodes, respectively, following established conjugation protocols while maintaining binding activity. For each adaptation, perform side-by-side comparisons with conventional immunofluorescence to ensure signal specificity is maintained during the modification process .

Develop appropriate controls specifically for each advanced imaging technique—include blank controls, isotype controls, and biological negative controls to distinguish specific signal from background or autofluorescence at the resolution limits of these techniques. Document both successful and failed adaptation attempts to guide future applications and contribute to the collective knowledge of antibody performance in advanced imaging contexts .

What are the considerations for using SPAC7D4.08 antibody in emerging single-cell analysis technologies?

Implementing SPAC7D4.08 antibody in single-cell analysis technologies requires special considerations to address the unique challenges of these methods. For single-cell proteomics approaches, validate antibody specificity and sensitivity at the substantially lower protein amounts present in individual cells compared to bulk samples. For drop-seq or microfluidic antibody-based detection, optimize concentration and binding kinetics to ensure efficient capture within the brief interaction times these platforms typically allow. When adapting for mass cytometry (CyTOF), conjugate the antibody with rare earth metals using established protocols and validate that metal labeling doesn't impair binding properties .

For spatial proteomics approaches like Digital Spatial Profiling or Multiplexed Ion Beam Imaging, validate antibody performance in fixed tissue contexts with attention to autofluorescence and non-specific binding that might confound single-cell resolution analyses. Consider competition assays with unlabeled antibody to confirm binding specificity in these highly sensitive systems. Across all single-cell applications, implement rigorous controls that account for the heightened impact of technical variability at the single-cell level .

Systematically optimize antibody concentration to identify the minimum concentration that provides reliable detection while minimizing background—this is particularly critical for single-cell analyses where signal-to-noise ratios are often challenging. Document titration experiments and optimization steps specifically for single-cell applications, as optimal conditions likely differ from bulk applications. This careful adaptation enables reliable incorporation of SPAC7D4.08 antibody into the rapidly expanding ecosystem of single-cell analysis technologies .