SENP2 Antibody, HRP conjugated

What is SENP2 and what cellular functions does it regulate?

SENP2 (Sentrin-specific protease 2) is a critical protease that catalyzes two essential functions in the SUMO (Small Ubiquitin-like Modifier) pathway. First, it hydrolyzes alpha-linked peptide bonds at the C-terminal end of SUMO propeptides (SUMO1, SUMO2, and SUMO3), which leads to the maturation of these proteins. Second, SENP2 deconjugates SUMO1, SUMO2, and SUMO3 from targeted proteins by cleaving epsilon-linked peptide bonds between the C-terminal glycine of mature SUMO proteins and the lysine epsilon-amino group of target proteins . These functions make SENP2 a critical regulator of protein SUMOylation, which affects protein localization, activity, and stability. Additionally, SENP2 may regulate β-catenin levels, thereby modulating the Wnt signaling pathway, and plays a role in processes like adipogenesis through its desumoylation activities .

What are the typical molecular characteristics of SENP2 antibodies?

SENP2 antibodies are typically developed with specific molecular characteristics that researchers should be aware of when designing experiments. The calculated molecular weight of SENP2 is approximately 68 kDa (67,855 Da specifically), though the observed molecular weight in experimental conditions typically falls between 60-68 kDa . Commercially available SENP2 antibodies are often produced in rabbit hosts and are available as polyclonal antibodies, such as the product described in search result #1 (29772-1-AP) and #4 (AP1232a) . These antibodies typically recognize specific epitopes, with some targeting the N-terminal region (such as amino acids 2-32) . Understanding these characteristics is essential for proper experimental planning and interpretation of results in Western blotting and other applications.

What applications are supported by SENP2 antibodies?

SENP2 antibodies support multiple research applications with varying dilution recommendations. For Western Blot (WB) applications, recommended dilutions typically range from 1:2000 to 1:10000, allowing researchers flexibility based on expression levels in their samples . For Immunohistochemistry (IHC) applications, dilutions between 1:500 and 1:2000 are generally recommended, with specific antigen retrieval protocols using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) . SENP2 antibodies have also been validated for ELISA applications . When using HRP-conjugated secondary antibodies for detection in Western blot experiments, researchers typically use dilutions around 1:2000, though this may vary by manufacturer . It's important to note that optimal dilutions should be determined empirically for each experimental system, as sample-dependent factors can influence performance .

What is the importance of proper storage for maintaining SENP2 antibody efficacy?

Proper storage of SENP2 antibodies is critical for maintaining their efficacy and extending their usable lifespan. These antibodies should be stored at -20°C for long-term preservation, where they remain stable for approximately one year after shipment . For antibodies in liquid form, they are typically provided in PBS with preservatives such as 0.02% sodium azide and 50% glycerol at pH 7.3 . When actively using the antibody, short-term storage at 2-8°C (refrigeration) is acceptable for up to two weeks . For some preparations, particularly with smaller volumes (20μL), manufacturers may include 0.1% BSA as a stabilizer . To minimize freeze-thaw cycles, which can degrade antibody performance, it is recommended to aliquot the antibody into smaller volumes before freezing, though some formulations note that aliquoting is unnecessary for -20°C storage . Always follow manufacturer-specific recommendations, as formulations may vary.

How can specificity of SENP2 antibodies be validated in experimental systems?

Validating SENP2 antibody specificity requires a multi-faceted approach. First, conduct positive control experiments using cell lines known to express SENP2, such as EC109, MKN-45, or SGC-7901 cells as mentioned in the product information . Implement both positive and negative controls by utilizing tissues or cell lines with known expression levels, including targeted knockdown experiments using siRNAs specific for SENP2, similar to the approach described in hepatocellular carcinoma research . Researchers should verify the antibody detects the expected molecular weight band (60-68 kDa) in Western blot applications . Cross-reactivity assessment is essential by testing the antibody against recombinant proteins of other SENP family members (SENP1, SENP3, etc.). For immunohistochemistry applications, include peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining . Additionally, comparing results from multiple antibodies targeting different epitopes of SENP2 can provide further confidence in specificity.

What are the critical factors for optimizing SENP2 detection in Western blot experiments?

Optimizing SENP2 detection in Western blot experiments involves careful attention to multiple technical factors. Sample preparation is crucial - use appropriate lysis buffers that preserve protein integrity while effectively extracting nuclear proteins like SENP2. Protein loading should be optimized (typically 20-50 μg total protein) with consistent loading across samples, verified with loading controls such as actin . For gel electrophoresis, 10% SDS-PAGE is commonly used for separating proteins in the SENP2 molecular weight range (60-68 kDa) . Transfer conditions should be optimized for proteins of this size, typically using PVDF or nitrocellulose membranes with appropriate transfer buffers and times . When blocking, use 5% non-fat milk in PBS or TBST (Tris-buffered saline with 0.1% Tween 20) for 1-2 hours at room temperature to minimize background . For primary antibody incubation, SENP2 antibodies perform optimally at dilutions between 1:2000-1:10000, typically incubated overnight at 4°C . When using HRP-conjugated secondary antibodies, incubate for 2 hours at room temperature with 1:2000 dilution . Finally, detection should use enhanced chemiluminescence systems with appropriate exposure times to avoid signal saturation .

How can researchers troubleshoot non-specific binding with SENP2 antibodies?

When troubleshooting non-specific binding with SENP2 antibodies, researchers should systematically address several key factors. First, optimize blocking conditions by testing different blocking agents (BSA, non-fat milk, commercial blockers) at various concentrations (3-5%) and durations (1-2 hours at room temperature) . Antibody dilution is critical - insufficient dilution often causes non-specific binding, so testing a broader range than the recommended 1:2000-1:10000 may be necessary . Washing protocols should be stringent, using TBST (0.1% Tween 20) with multiple (4-5) washes of 5-10 minutes each after both primary and secondary antibody incubations . If background persists, consider adding 0.1-0.5% BSA to antibody dilution buffers to reduce non-specific interactions. For cross-reactivity issues, pre-absorb the antibody with cell/tissue lysates from non-expressing samples or use more specific monoclonal antibodies when available. If membrane-wide background occurs, verify the quality and freshness of blocking agents and wash buffers. For IHC applications, optimize antigen retrieval methods, comparing citrate buffer (pH 6.0) and TE buffer (pH 9.0) as suggested in product information .

What experimental design considerations are important when studying SENP2 in cancer research?

When designing experiments to study SENP2 in cancer research, several critical considerations must be addressed. First, selection of appropriate cell models is essential - research has shown that SENP2 expression varies significantly across cancer types, with documented downregulation in hepatocellular carcinoma tissues compared to adjacent normal tissues . Researchers should incorporate multiple cell lines representing both normal and cancerous states for comparative analyses. Knockdown and overexpression experiments are powerful approaches, as demonstrated in the HepG2 hepatocellular carcinoma model where SENP2 overexpression suppressed cell growth while SENP2 silencing promoted proliferation and colony formation . When analyzing clinical samples, paired tumor and adjacent normal tissue comparisons provide the most informative data, with both mRNA (quantitative PCR) and protein (Western blot) analyses recommended for comprehensive profiling . Functional assays should include proliferation, colony formation, and pathway-specific readouts, particularly focusing on the β-catenin/Wnt pathway which has been implicated in SENP2's tumor-suppressive functions . For mechanistic studies, design experiments that distinguish between SENP2's effects on specific SUMO isoforms (SUMO1 vs. SUMO2/3), as these may have distinct consequences for target protein function .

How can researchers effectively analyze SENP2-mediated effects on protein SUMOylation?

Analyzing SENP2-mediated effects on protein SUMOylation requires sophisticated experimental approaches. Researchers should implement in vitro deSUMOylation assays using purified components, including SENP2, SUMO-conjugated substrates, and appropriate buffers and reaction conditions to directly measure SENP2's enzymatic activity. For cellular studies, comparative SUMOylation profiling using SENP2 overexpression and knockdown/knockout models provides valuable insights into substrate specificity . Western blot analyses should include detection of both free SUMO proteins and SUMO-conjugated target proteins, utilizing specific antibodies against SUMO1 and SUMO2/3 as described in the literature . When analyzing specific target proteins, such as amyloid precursor protein (APP) or β-catenin, researchers should employ co-immunoprecipitation approaches coupled with SUMOylation-specific antibodies to isolate and identify SUMOylated forms . Mass spectrometry-based proteomics offers the most comprehensive approach for identifying SENP2 substrates on a global scale, particularly when combined with SUMO remnant antibodies that recognize the diglycine motif left after deSUMOylation. Statistical analysis should incorporate appropriate controls and quantification methods, with significance typically represented at different thresholds (p < 0.05, p < 0.01, or p < 0.001) as documented in SENP2 research publications .

What are the advantages of using HRP-conjugated antibodies for SENP2 detection?

HRP-conjugated antibodies offer several significant advantages for SENP2 detection in research applications. Direct HRP conjugation eliminates the need for secondary antibody incubation steps, reducing experimental time by approximately 2-3 hours and minimizing potential sources of cross-reactivity and background . This approach is particularly valuable in multiplex detection systems where antibodies from the same host species might be used simultaneously. HRP-conjugated antibodies provide enhanced sensitivity through enzymatic signal amplification, potentially detecting lower abundance proteins like SENP2 in samples where expression might be reduced, such as in hepatocellular carcinoma tissues where SENP2 has been shown to be downregulated . The quantitative linearity of chemiluminescent detection using HRP systems makes these conjugates ideal for comparative studies analyzing SENP2 expression levels across different experimental conditions or tissue samples. Additionally, HRP-conjugated antibodies are compatible with numerous detection systems, including chemiluminescence, chromogenic substrates, and tyramide signal amplification, providing flexibility in experimental design based on available equipment and sensitivity requirements .

How should researchers optimize chemiluminescent detection with HRP-conjugated SENP2 antibodies?

Optimizing chemiluminescent detection with HRP-conjugated SENP2 antibodies requires attention to several critical parameters. First, substrate selection is crucial - enhanced chemiluminescence (ECL) substrates vary in sensitivity, with standard ECL appropriate for abundant proteins and enhanced systems (such as the Clarity Western ECL Substrate mentioned in the literature) necessary for detecting lower-expression proteins like SENP2 in certain tissues . Exposure time optimization is essential - begin with short exposures (30 seconds) and incrementally increase up to 5 minutes to determine the optimal signal-to-noise ratio, using an image analyzer system similar to the Pxi or Syngene systems referenced in the research . Antibody dilution should be carefully titrated, typically starting with the manufacturer's recommendation (often 1:2000-1:10000 for primary antibodies) and adjusting based on signal intensity . The membrane handling technique impacts detection quality - after antibody incubations, ensure thorough washing (4-5 times for 5 minutes each) in TBST to remove unbound antibody . For quantitative analysis, use appropriate software (such as Gene Tools mentioned in the research) and normalize SENP2 signals to loading controls like actin . When troubleshooting weak signals, consider longer primary antibody incubation (overnight at 4°C), reduced washing stringency, or switching to a more sensitive ECL substrate system.

What controls should be included when using HRP-conjugated antibodies in SENP2 research?

When using HRP-conjugated antibodies for SENP2 research, a comprehensive set of controls is essential for ensuring experimental validity. Loading controls are fundamental - researchers should include housekeeping protein detection (such as actin at 1:20000 dilution) to normalize for variations in protein loading across samples . Positive control samples from cell lines with confirmed SENP2 expression (such as EC109, MKN-45, or SGC-7901 cells) should be included to verify antibody functionality . For tissue-based studies, normal pancreatic tissue has been validated for SENP2 detection in IHC applications . Negative controls should include SENP2-knockdown samples generated using validated siRNAs, with research showing that SENP2-specific siRNAs can effectively reduce endogenous SENP2 expression in cell models like HepG2 . Antibody-specific controls should include primary antibody omission controls to assess secondary antibody specificity and background, and isotype controls (normal rabbit IgG at the same concentration as the primary antibody) to distinguish specific from non-specific binding . For HRP enzyme activity verification, include a positive control protein at known concentration detected with the same detection system. When conducting quantitative analysis, include a standard curve using recombinant SENP2 protein at known concentrations to ensure signal linearity within the experimental detection range.

How can SENP2 antibodies be used to investigate the interplay between SENP1 and SENP2 in disease models?

Investigating the interplay between SENP1 and SENP2 using antibodies requires sophisticated experimental design. Differential expression analysis is fundamental - researchers should utilize specific antibodies against both SENP1 (such as NB100-56405, Novusbio) and SENP2 (such as AP1232, Abgent or 29772-1-AP, Proteintech) in parallel Western blots or multiplexed immunofluorescence to compare their expression patterns across tissues or disease states . This approach has revealed that both SENP1 and SENP2 can induce de-SUMOylation of amyloid precursor protein (APP), but their expression patterns differ in disease models . Co-immunoprecipitation experiments using SENP2 antibodies followed by SENP1 detection (or vice versa) can identify potential physical interactions or complexes between these proteases. For functional analysis, researchers should employ selective knockdown approaches targeting either SENP1 or SENP2 individually and in combination, then assess effects on global SUMOylation patterns and specific substrate modifications using SUMO1 and SUMO2/3-specific antibodies (1:2000 dilution, Cell Signaling Technology) . Quantitative PCR reveals different regulatory patterns, as SENP1 expression may increase in certain disease models while SENP2 remains unchanged . Cell-specific expression patterns should be assessed through immunohistochemistry or immunofluorescence in tissue sections using the validated dilution ranges (1:500-1:2000 for IHC) . This comprehensive approach enables researchers to distinguish unique and overlapping functions of these related proteases in disease pathogenesis.

What methodological approaches can differentiate between SENP2's effects on different SUMO isoforms?

Differentiating between SENP2's effects on different SUMO isoforms requires specialized methodological approaches. Isoform-specific antibody detection is fundamental - researchers should employ distinct antibodies that specifically recognize SUMO1 versus SUMO2/3 (such as those from Cell Signaling Technology, used at 1:2000 dilution) in Western blot analyses of samples with modulated SENP2 expression . In vitro enzymatic assays can be designed using recombinant SENP2 and individually tagged SUMO isoforms (SUMO1, SUMO2, and SUMO3) to directly measure catalytic preferences through kinetic analyses. For cellular studies, researchers can express individual SUMO isoforms tagged with different epitopes or fluorescent markers, then assess how SENP2 overexpression or knockdown differentially affects their conjugation patterns. Substrate-specific analyses should examine how SENP2 modulation impacts the SUMOylation status of known targets by specific SUMO isoforms - for example, β-catenin has been identified as a SENP2-regulated protein in hepatocellular carcinoma models . Quantitative proteomics approaches using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with SUMO-remnant antibodies can provide global profiling of SENP2's isoform preferences. Localization studies using immunofluorescence microscopy with isoform-specific SUMO antibodies can reveal whether SENP2 preferentially affects nuclear, cytoplasmic, or other subcellular pools of different SUMO conjugates.

How should researchers design experiments to study SENP2's role in the β-catenin pathway?

Designing experiments to study SENP2's role in the β-catenin pathway requires systematic approaches to establish causal relationships. Expression correlation studies should analyze SENP2 and β-catenin levels in paired samples using Western blotting with specific antibodies for each protein, as demonstrated in hepatocellular carcinoma research where SENP2 downregulation was associated with β-catenin regulation . Genetic modulation experiments should include both overexpression of Flag-tagged SENP2 and silencing of endogenous SENP2 using validated siRNAs (with siRNA #2 showing more efficient knockdown in previous studies), followed by assessment of β-catenin protein levels, stability, and transcriptional activity . For mechanistic investigations, researchers should perform co-immunoprecipitation experiments using either SENP2 or β-catenin antibodies to detect physical interactions, combined with in vitro deSUMOylation assays to determine if β-catenin is a direct SENP2 substrate. Functional readouts must include both β-catenin protein stability measurements and assessment of downstream Wnt pathway activity through reporter assays (TOPFlash) and target gene expression analysis. Cellular phenotype correlation is essential - researchers should connect SENP2-mediated changes in β-catenin to functional outcomes such as cell proliferation and colony formation, using assays similar to those that demonstrated SENP2 overexpression suppressed HepG2 cell growth while SENP2 silencing promoted it . Tissue-specific analyses in clinical samples should examine the correlation between SENP2 expression, β-catenin levels, and pathological features in diseases where the Wnt pathway is implicated.

What considerations are important when using SENP2 antibodies for detecting post-translational modifications?

When using SENP2 antibodies to detect post-translational modifications, researchers must address several technical considerations. For detecting SUMOylated proteins, denaturing lysis conditions are essential to inactivate endogenous SENP proteases and preserve SUMO modifications - typically using 1% SDS or 8M urea with SUMO protease inhibitors (such as N-ethylmaleimide at 20mM) . Antibody selection should include both SENP2-specific antibodies (such as the polyclonal 29772-1-AP or AP1232) and modification-specific antibodies (such as anti-SUMO1 and anti-SUMO2/3 antibodies) to fully characterize the system . Enrichment strategies improve detection sensitivity - researchers should consider immunoprecipitation of either the target protein or the modification itself before Western blotting. For technical optimization, extended gel electrophoresis (using gradient gels) improves separation of modified proteins, which typically show higher molecular weights than unmodified forms. Control experiments must include known SENP2 substrates as positive controls and SENP2 enzyme inhibition (genetic or chemical) to confirm modification-specific signals. When performing mass spectrometry analyses, researchers should implement targeted approaches to identify specific modification sites, such as enriching for diglycine remnant peptides that indicate SUMO attachment points. For complex analyses involving multiple modifications, consider combinatorial approaches using antibodies against different modifications sequentially or in parallel to assess potential crosstalk between SUMOylation and other modifications like phosphorylation or ubiquitination.

How should researchers quantify and statistically analyze SENP2 expression data?

Quantification and statistical analysis of SENP2 expression data requires rigorous methodological approaches. For Western blot quantification, researchers should use densitometry software (such as Gene Tools mentioned in the hepatocellular carcinoma study) to measure band intensities, with normalization to loading controls such as actin . When comparing SENP2 expression between samples, such as tumor versus adjacent normal tissues, calculate relative expression levels and present them as fold-changes, as demonstrated in the study of 25 hepatocellular carcinoma patients . For statistical significance testing, apply appropriate tests based on data distribution - typically Student's t-tests for pairwise comparisons with significance thresholds clearly indicated (* for P < 0.05, ** for P < 0.01, *** for P < 0.001) . When analyzing quantitative PCR data for SENP2 mRNA levels, utilize the comparative CT method (2^-ΔΔCT) with appropriate reference genes for normalization. For immunohistochemistry quantification, implement standardized scoring systems incorporating both staining intensity and percentage of positive cells, with multiple independent observers to ensure reliability. In functional experiments examining SENP2's effects on cellular phenotypes, conduct at least three independent experiments and present data as mean ± standard deviation (SD) . For clinical correlations, use appropriate multivariate analyses to control for confounding variables when associating SENP2 expression with clinical parameters or outcomes. Sample size determination should be based on power calculations to ensure sufficient statistical power, particularly for human tissue studies where SENP2 expression has been shown to vary significantly across individuals .

What are the common pitfalls in interpreting SENP2 antibody experimental results?

When interpreting SENP2 antibody experimental results, researchers should be vigilant about several common pitfalls. Antibody cross-reactivity with other SENP family members (particularly SENP1, which shares functional similarity with SENP2) can lead to misinterpretation of specificity - always validate antibody specificity using knockout/knockdown controls or peptide competition assays . Signal interpretation errors can occur when misidentifying the correct molecular weight band - while SENP2's calculated molecular weight is approximately 68 kDa, the observed molecular weight in experimental systems typically ranges from 60-68 kDa . Context-dependent expression variations can complicate comparisons across experimental systems - SENP2 has been shown to be downregulated in specific cancers like hepatocellular carcinoma, but expression patterns may differ in other tissues or disease states . Quantification biases often arise when Western blot exposures are saturated - ensure linear detection range for accurate densitometry. Technical variability in immunohistochemistry can result from different antigen retrieval methods - compare results using both TE buffer (pH 9.0) and citrate buffer (pH 6.0) as suggested in product information . Attribution errors occur when assigning phenotypic changes directly to SENP2 without considering its numerous substrates - SENP2 affects multiple proteins including β-catenin, and pathway analyses should be comprehensive . For inconsistent results across detection methods (protein versus mRNA), consider post-transcriptional regulation mechanisms rather than assuming technical error. When comparing results with published literature, account for differences in antibody sources, cell types, and experimental conditions.

How can researchers effectively validate novel SENP2 substrates using antibody-based approaches?

Validating novel SENP2 substrates using antibody-based approaches requires a multi-layered experimental strategy. In vitro deSUMOylation assays provide direct evidence - incubate purified, SUMOylated candidate proteins with recombinant SENP2 enzyme, then detect SUMOylation status changes using Western blot with substrate-specific and SUMO-specific antibodies (anti-SUMO1 and anti-SUMO2/3 at 1:2000 dilution) . For cellular validation, implement bidirectional genetic approaches - both SENP2 overexpression and knockdown/knockout - and examine corresponding changes in substrate SUMOylation status. Co-immunoprecipitation experiments should demonstrate physical interaction between SENP2 and the candidate substrate, optimally under conditions that preserve transient enzyme-substrate interactions. Site-directed mutagenesis of predicted SUMOylation sites (lysine residues) in the candidate substrate, followed by assessment of SENP2 sensitivity, provides strong evidence for direct modification. For endogenous interaction confirmation, proximity ligation assays (PLA) using specific antibodies against both SENP2 and the candidate substrate can visualize interactions in situ. Functional consequence validation is essential - demonstrate that SENP2-mediated deSUMOylation alters substrate properties such as localization, stability, or activity, similar to how SENP2 has been shown to affect β-catenin stability in hepatocellular carcinoma models . Domain mapping experiments using truncated versions of both SENP2 and the substrate can identify specific interaction regions. Finally, correlation analyses in relevant tissue samples should assess whether SENP2 expression levels inversely correlate with substrate SUMOylation status in physiological or pathological contexts.

How can SENP2 antibodies be applied in multiplex immunofluorescence studies?

Applying SENP2 antibodies in multiplex immunofluorescence studies requires strategic planning and technical optimization. Antibody selection is critical - choose SENP2 antibodies validated for immunofluorescence applications, preferably those raised in host species different from other target antibodies to minimize cross-reactivity . Sequential antibody application protocols should be designed with careful consideration of epitope availability and potential blocking effects - typically beginning with the lowest abundance target (often SENP2) using signal amplification methods if necessary. For spectrally distinct detection, directly conjugate SENP2 antibodies with fluorophores in non-overlapping emission spectra or use secondary antibodies with minimal spectral overlap. Tyramide signal amplification can enhance detection sensitivity for low-abundance proteins like SENP2, particularly in tissues where expression is downregulated such as hepatocellular carcinoma . Control experiments must include single-stain controls for each antibody to confirm specificity and absence of spectral bleed-through, as well as biological controls (SENP2 knockdown/overexpression) to validate signal specificity. For subcellular localization studies, combine SENP2 antibody staining with markers for cellular compartments (nucleus, cytoplasm, organelles) to precisely map SENP2 distribution. Image acquisition parameters should be optimized for each fluorophore, with linear detection ranges established using calibration standards. Automated multispectral imaging platforms can improve signal separation and enable quantitative analysis of SENP2 co-localization with potential substrates or interacting proteins across different subcellular compartments and cell types within complex tissues.

What approaches are recommended for developing CRISPR/Cas9 knockout validation systems for SENP2 antibodies?

Developing CRISPR/Cas9 knockout validation systems for SENP2 antibodies requires comprehensive strategic planning. Guide RNA design should target early exons of the SENP2 gene to maximize knockout efficiency, with multiple guides designed and tested to identify optimal targeting sequences. For cellular models, select relevant cell lines that express detectable levels of endogenous SENP2, such as those identified in antibody validation studies (EC109, MKN-45, SGC-7901) or hepatocellular carcinoma cell lines like HepG2 where SENP2 function has been characterized . Knockout verification must be multi-level - genomic validation through sequencing of the targeted region, transcript analysis using RT-qPCR, and most critically, protein-level validation using the specific SENP2 antibodies being assessed at recommended dilutions (1:2000-1:10000 for Western blot) . Control systems are essential - maintain wild-type parental cells alongside the knockout lines and include positive controls for antibody functionality. For comprehensive validation, generate multiple independent knockout clones to control for clonal variation and off-target effects. Rescue experiments provide the strongest validation - re-express SENP2 in knockout cells to demonstrate restoration of antibody signal. Functional validation should assess previously characterized SENP2-dependent phenotypes, such as effects on cell proliferation and colony formation in HepG2 cells or β-catenin stability . For antibodies intended for specific applications, perform validation in those precise contexts - for example, validate antibodies intended for IHC by performing immunostaining on fixed, paraffin-embedded sections of wild-type versus knockout cells at the recommended dilutions (1:500-1:2000) .