SPCC70.10 Antibody

What is the SPCC70.10 antibody and what is its target specificity?

The SPCC70.10 antibody is a research-grade antibody product available from manufacturers like CUSABIO-WUHAN HUAMEI BIOTECH Co., Ltd. While specific target information for this particular antibody must be confirmed through manufacturer documentation, antibodies in this class are typically designed to recognize specific protein targets with high specificity.

To determine the target specificity of any antibody including SPCC70.10:

• Review the manufacturer's documentation for the specific epitope information

• Perform Western blot analysis against known positive and negative controls

• Consider knockout validation where the target protein is absent (CRISPR/Cas9)

• Evaluate cross-reactivity through immunoprecipitation followed by mass spectrometry

Thorough validation is essential as studies have shown that many commercially available antibodies may not perform as advertised, with one study finding only 48% of 3,313 antibodies recognizing their intended protein in Western blotting applications .

Which experimental applications is SPCC70.10 antibody validated for?

When considering applications for antibodies like SPCC70.10, researchers should:

• Check the manufacturer's validation data for recommended applications

• Review application-specific validation data for:

  • Western blot (WB)

  • Immunohistochemistry (IHC)

  • Immunocytochemistry/Immunofluorescence (ICC/IF)

  • Flow cytometry

  • Immunoprecipitation (IP)

It's important to note that validation for one application does not guarantee performance in another. For example, antibodies that perform well in Western blot may not work in IHC due to differences in protein conformation and processing . Always validate the antibody for your specific application before use in critical experiments.

How should I validate SPCC70.10 antibody for my specific application?

Validation should follow a multi-step approach:

• Initial testing with positive controls: Use samples known to express the target protein

• Negative control verification: Test in samples lacking target expression

• Application-specific validation:

  • For Western blot: Check for single band at expected molecular weight

  • For IHC/ICC: Confirm proper subcellular localization

  • For IP: Verify pull-down of target protein through mass spectrometry

The International Working Group for Antibody Validation recommends multiple validation strategies including :

• Genetic strategies (knockout/knockdown)

• Orthogonal strategies (comparing with other measurement techniques)

• Independent antibody strategies (using a second antibody against a different epitope)

• Expression of tagged proteins

• Immunocapture followed by mass spectrometry

How can I distinguish between true signal and non-specific binding when using SPCC70.10 antibody?

Non-specific binding remains one of the most challenging aspects of antibody-based research. To distinguish true from non-specific signals:

• Peptide competition assays: Pre-incubate antibody with excess synthetic peptide containing the epitope sequence; true signals should be blocked

• Knockout/knockdown controls: Use genetic techniques to create samples lacking the target

• Signal correlation analysis: True signals should correlate with known expression patterns of the target

• Titration experiments: Specific signals typically show dose-dependent changes with antibody concentration, whereas non-specific signals may not

• Multiple antibody validation: Use alternative antibodies recognizing different epitopes of the same protein

For phosphorylation-specific antibodies, additional controls are needed including:

• Phosphatase treatment of samples (should eliminate signal)

• Stimulation/inhibition experiments to modulate phosphorylation status

• Mutation of the phosphorylation site in recombinant proteins

What strategies can address batch-to-batch variability when using SPCC70.10 antibody in longitudinal studies?

Batch-to-batch variability presents a significant challenge for longitudinal studies. To address this:

• Strategic purchasing: Buy sufficient quantity of a single lot for the entire study

• Lot validation: Thoroughly validate each new lot against previous lots using identical samples

• Reference standards: Maintain reference samples to test each new lot

• Internal controls: Include consistent positive and negative controls in every experiment

• Normalization strategies: Develop robust normalization methods based on housekeeping proteins

• Documentation: Maintain detailed records of lot numbers, validation data, and experimental conditions

Consider using recombinant antibodies when available, as these typically show less batch-to-batch variation compared to monoclonal and polyclonal antibodies. A comprehensive study by Ayoubi et al. found that recombinant antibodies performed better across multiple tests compared to traditional antibody types .

How should I troubleshoot conflicting results between SPCC70.10 antibody and other detection methods?

When antibody results conflict with other methods:

• Evaluate antibody validation: Reassess the antibody's specificity using knockout controls or peptide competition

• Consider protein modifications: Post-translational modifications may affect antibody binding

• Assess protein conformation: Native vs. denatured states can influence epitope accessibility

• Check for splice variants: Different detection methods may recognize different isoforms

• Examine subcellular localization: Proteins may be sequestered in different cellular compartments

• Review experimental conditions: Buffer compositions, fixation methods, and antigen retrieval techniques can all affect results

Systematic analysis of discrepancies through methodological variation can provide insights into the nature of conflicts. Document all variables including:

What factors influence epitope accessibility when using SPCC70.10 antibody in different techniques?

Epitope accessibility varies significantly across techniques:

• Protein conformation factors:

  • Secondary/tertiary structure may mask epitopes in native conditions

  • Denaturation may expose or destroy epitopes depending on their nature

  • Oligomerization or protein-protein interactions can block binding sites

• Sample preparation effects:

  • Fixation methods (formaldehyde, methanol, acetone) differentially affect epitope structure

  • Cross-linking can permanently mask epitopes

  • Antigen retrieval techniques may be required to recover epitopes after fixation

• Buffer conditions impact:

  • pH affects protein charge and conformation

  • Detergent types and concentrations influence protein solubility and structure

  • Salt concentration affects electrostatic interactions

• Post-translational modifications:

  • Phosphorylation, glycosylation, or ubiquitination may mask epitopes

  • Modifications near the epitope may prevent antibody binding

Research from antibody validation studies shows that epitope accessibility is a primary reason why antibodies may work in Western blot but fail in immunohistochemistry or vice versa .

How should I design proper controls when using SPCC70.10 antibody in multicolor immunofluorescence?

Multicolor immunofluorescence requires rigorous controls:

• Single-color controls: Stain separate samples with each primary-secondary combination to assess spectral overlap

• Isotype controls: Use matched isotype antibodies to evaluate non-specific binding

• Blocking peptide controls: Competition with the immunizing peptide to confirm specificity

• Secondary-only controls: Omit primary antibody to assess secondary antibody background

• Knockout/negative controls: Include samples lacking the target protein

• Positive controls: Include samples with known expression of target proteins

For advanced multiplexing, consider these additional controls:

What are the methodological considerations for quantitative analysis using SPCC70.10 antibody?

Quantitative analysis requires careful methodological considerations:

• Standard curve establishment:

  • Use purified recombinant proteins of known concentrations

  • Create a dilution series to establish linear range

  • Determine lower and upper limits of detection

• Signal normalization strategies:

  • Normalize to housekeeping proteins (for Western blot)

  • Use ratio to DNA content (for flow cytometry)

  • Implement cell area normalization (for imaging)

• Technical replication:

  • Perform technical triplicates at minimum

  • Assess coefficient of variation between replicates (<15% is typically acceptable)

• Controls for quantification:

  • Include calibrator samples in each experiment

  • Run inter-assay controls to normalize between experiments

• Image analysis parameters:

  • Standardize exposure settings

  • Define consistent thresholding methods

  • Document all image processing steps

Studies have demonstrated that application-specific validation is essential for quantitative analyses, as antibody performance characteristics like dynamic range and linearity vary between applications .

How can I optimize fixation and permeabilization protocols for SPCC70.10 antibody in immunocytochemistry?

Optimization requires systematic testing of conditions:

• Fixation method screening:

  • Test paraformaldehyde (1-4%) for structure preservation

  • Try methanol for cytoskeletal proteins

  • Evaluate glutaraldehyde for membrane proteins

  • Consider combined protocols for challenging targets

• Fixation duration optimization:

  • Short fixation (5-10 min) for surface proteins

  • Standard fixation (15-20 min) for most applications

  • Extended fixation (30+ min) for some nuclear proteins

• Permeabilization agent comparison:

  • Triton X-100 (0.1-0.5%) for nuclear proteins

  • Saponin (0.1-0.3%) for reversible membrane permeabilization

  • Digitonin (10-50 μg/ml) for selective plasma membrane permeabilization

• Antigen retrieval evaluation:

  • Heat-induced epitope retrieval (citrate or EDTA buffers)

  • Enzymatic retrieval (proteinase K, trypsin)

  • pH variation (pH 6.0, 8.0, or 9.0)

The optimal protocol will depend on the specific target's subcellular localization and the epitope recognized by SPCC70.10. Create a matrix of conditions and evaluate signal-to-noise ratio for each:

How do different sample preparation methods affect SPCC70.10 antibody performance in immunoblotting?

Sample preparation significantly impacts antibody performance:

• Lysis buffer composition effects:

  • RIPA buffer: Good for membrane and nuclear proteins

  • NP-40/Triton buffers: Gentler for cytoplasmic and membrane proteins

  • SDS-based buffers: Harsh but effective for difficult proteins

  • Urea-based buffers: For highly insoluble proteins

• Protein denaturation considerations:

  • Boiling duration (1-10 minutes)

  • Temperature variations (37°C, 65°C, 95°C)

  • Reducing agent concentration (DTT or β-mercaptoethanol)

• Sample handling impact:

  • Freeze-thaw cycles (minimize to <3)

  • Storage temperature (-20°C vs. -80°C)

  • Protease/phosphatase inhibitor inclusion

• Loading control selection:

  • Choose loading controls appropriate for your experimental conditions

  • Consider that treatments may affect "housekeeping" proteins

Research on antibody validation has shown that sample preparation methods can dramatically influence detection sensitivity and specificity. For instance, heat shock proteins like HSP70 may show different patterns depending on sample preparation methods due to their chaperone functions and stress-responsive nature .

What are the molecular mechanisms behind false-positive signals with antibodies like SPCC70.10?

Understanding false-positive mechanisms helps in troubleshooting:

• Cross-reactivity sources:

  • Shared epitopes between related proteins

  • Common structural motifs recognized by antibody

  • Post-translational modifications present on multiple proteins

• Non-specific binding mechanisms:

  • Fc receptor interactions on cells

  • Hydrophobic interactions with denatured proteins

  • Charge-based interactions with highly charged proteins

• Technical artifacts:

  • Secondary antibody cross-reactivity

  • Endogenous peroxidase or phosphatase activity

  • Autofluorescence in specific wavelengths

• Tissue-specific considerations:

  • Endogenous biotin in certain tissues

  • High background in adipose or liver tissue

  • Necrotic tissue binding antibodies non-specifically

Studies have found that even highly specific monoclonal antibodies can produce false positive signals due to these mechanisms, emphasizing the importance of proper controls and validation strategies .

How can I adjust protocols when switching between monoclonal and polyclonal versions of antibodies against the same target?

Transitioning between antibody types requires protocol adjustments:

• Concentration optimization:

  • Polyclonals typically used at 1:200-1:1000 dilutions

  • Monoclonals often effective at 1:1000-1:5000 dilutions

  • Titration experiments essential for each new antibody

• Incubation conditions:

  • Polyclonals may work with shorter incubation times

  • Monoclonals sometimes benefit from longer/overnight incubations

  • Temperature optimization (4°C, room temperature, 37°C)

• Blocking strategy differences:

  • Polyclonals may require stronger blocking (5-10% serum)

  • Monoclonals sometimes work with reduced blocking (1-3% BSA)

  • Species matching considerations for blocking serum

• Signal detection adjustments:

  • Polyclonals generally produce stronger signals requiring shorter exposure

  • Monoclonals may need signal enhancement techniques

  • Secondary antibody concentration adjustments

Research has shown that monoclonal antibodies typically offer higher reproducibility but potentially lower sensitivity compared to polyclonals, which may detect multiple epitopes but have higher batch variation .

How can SPCC70.10 antibody be used in combination with other techniques for protein-protein interaction studies?

Integrating antibodies with complementary techniques enhances protein interaction studies:

• Co-immunoprecipitation enhancements:

  • Sequential immunoprecipitation to identify multiprotein complexes

  • Crosslinking prior to immunoprecipitation for transient interactions

  • Comparison of different lysis conditions to preserve different interaction types

• Proximity ligation assay (PLA) implementation:

  • Combine SPCC70.10 with antibodies against potential interaction partners

  • Optimize probe concentration and incubation times

  • Include appropriate controls (single antibody, non-interacting protein pairs)

• FRET/BRET analysis integration:

  • Use antibodies to validate interactions observed with fluorescent/bioluminescent tags

  • Compare endogenous interactions (antibody-detected) with tagged protein results

• Mass spectrometry validation:

  • Use antibody immunoprecipitation followed by MS to identify novel interactions

  • Validate MS-identified interactions with reciprocal immunoprecipitation

What considerations are important when using SPCC70.10 antibody across different species or tissues?

Cross-species and cross-tissue applications require careful validation:

• Epitope conservation analysis:

  • Perform sequence alignment of the epitope region across species

  • Predict potential differences in post-translational modifications

  • Consider evolutionary conservation of protein structure

• Tissue-specific validation steps:

  • Test antibody in multiple tissues independently

  • Adjust protocols for tissue-specific factors (fixation time, antigen retrieval)

  • Account for different expression levels across tissues

• Species-specific optimization:

  • Adjust antibody concentration for each species

  • Modify blocking conditions to reduce background

  • Consider species-matched secondary antibodies

• Potential confounding factors:

  • Endogenous biotin in certain tissues (liver, kidney)

  • Autofluorescence in specific tissues (brain, liver)

  • High background in adipose tissue

Research has shown that antibody performance can vary dramatically across species even with highly conserved proteins. For heat shock proteins like HSP70, epitope accessibility may differ between species due to interaction partners and conformational states .

How can SPCC70.10 antibody be incorporated into high-throughput or automated workflows?

Implementing antibodies in high-throughput systems requires:

• Assay miniaturization strategies:

  • Optimize antibody concentration for microwell formats

  • Determine minimum sample volume requirements

  • Establish signal detection limits in reduced volumes

• Automation compatibility assessment:

  • Test antibody stability in automated handling conditions

  • Optimize incubation times for robotic workflows

  • Validate performance with automated washing systems

• Batch consistency requirements:

  • Implement quality control metrics for each antibody batch

  • Create standard operating procedures for lot testing

  • Maintain reference samples for inter-batch calibration

• Multiplexing capabilities:

  • Evaluate antibody performance in multiplexed detection systems

  • Test for cross-reactivity with other antibodies in the panel

  • Optimize signal separation for multiple readouts

• Data analysis automation:

  • Develop standardized analysis pipelines

  • Implement quality control metrics in the analysis workflow

  • Validate automated vs. manual analysis concordance