Antibody Validation with ShGE™ Platform: Overcoming the Limitations of Single Application Validation

Gene knock-out (KO) and knock-down (KD) technologies are internationally recognized as the gold standard methods for validating antibody specificity. KO completely removes or disrupts the target gene at the DNA level, while KD reduces target gene expression at the RNA level. Both approaches eliminate or silence the endogenous target protein to assess antibody binding and validate specificity.

Although KO can thoroughly remove the target gene-encoded protein, it is limited to approximately 6,000 human genes; GenuIN Biotech’s ShGE™ gene-silencing platform (ShGE™ Platform) performs KD at the mRNA level, opening up the possibility to cover all 20,000 protein-coding human genes. KD reduces mRNA and protein expression of the target gene in specific cell lines, which can serve as functional knockout (negative control) samples in experiments. If an antibody shows a signal in the wild-type sample but the signal disappears or is greatly reduced in the KD sample, the antibody is considered specific.

After KD of the target gene and its corresponding protein in cells, validation of expression levels of the target gene is typically done through quantitative real-time polymerase chain reaction/quantitative polymerase chain reaction (qRT-PCR/qPCR), Western blotting (WB), immunocytochemistry (ICC), or flow cytometry (FCM). Limiting the validation to any one application has its drawbacks. For example, qPCR measures only mRNA levels and may overestimate KD efficiency. WB can suffer from factors such as recognizing homologous proteins, degradation fragments, or post-translational variants, potentially leading to misinterpretation of results. Matching only by WB band size cannot eliminate these possibilities. Therefore, the ShGE™ platform employs not only qPCR and WB, but also ICC, and FCM in a multi-applicational validation approach to avoid the limitations of relying on a single method.

1  ShGE™ Platform for Validating Antibody Specificity

GenuIN Biotech's ShGE™ platform integrates, optimizes, and innovates upon traditional KD workflows. It uses lentivirus-mediated shRNA to silence specific mRNAs, leading to the disappearance or significant downregulation of the corresponding protein signa. Our process is simplified from 16 steps to 4, reduces the time from 6 months to 2 weeks, increases the success rate from 40% to over 70%, and enables 500 KDs per week, covering targets across the entire human genome. Thus, the ShGE™ platform achieves high-throughput, low-cost, and genome-wide antibody specificity validation—better meeting market demands for antibodies with high specificity and reproducibility.

By testing over 10,000 antibodies, our platform has revealed that fewer than 20% of commercially available monoclonal antibodies and only 1% of polyclonal antibodies are truly specific. Additionally, among antibodies considered highly specific based on WB results, only 70% showed specificity in ICC or FCM. Therefore, the ShGE™ platform not only exposes the issues of the antibody market, but is a much needed technology to provide tested and high quality reagents for researchers and the biotech industry.

2 ShGE™ Platform’s "Multi-Applicational Validation Strategy" to Resolve Conflicting Results

To ensure the reliability of KD experiment results, a multi-technology strategy should be used for comprehensive analysis of target protein signals:First, use FCM to select KD cell populations with high silencing efficiency to avoid false-negative WB results caused by WT cell contamination;

Figure 1. Multi-dimensional Validation Workflow Diagram

Then, apply ICC to sorted cells to examine subcellular localization changes such as nuclear aggregation or membrane disappearance, revealing potential compensatory mechanisms. When ICC/FCM indicates altered localization but not total expression change of the target protein, WB should be used to eliminate the possibility of antibody cross-reactivity (Figure 1). The reliability of multi-dimensional strategies depends on the consistency and specificity of antibodies—only those validated across ICC/FCM/WB by the ShGE™ platform can ensure cross-technology data confirmation and prevent any errors that may arise from isolated experiments.

2.1  Multi-Dimensional Validation Case Study of KD Experiments on the ShGE™ Platform

Figure 2 shows the expression of CREB1 antibody validated by Western blot (WB), flow cytometry (FCM), and immunocytochemistry (ICC) in wild-type (WT) and CREB1 knockdown (KD) HeLa cell lines.

As seen in Figure 2A, the WB band for CREB1 disappears completely in KD cells, showing a 100% reduction compared to WT.

However, in FCM (Figure 2B) and ICC (Figure 2C), the signal in WT and KD HeLa cells shows no significant difference.

Why do some KD cell lines show successful knockdown results in qPCR and WB, yet exhibit contradictory results in phenotype-based methods such as ICC or FCM?

This may be due to WB detecting degradation fragments (or homologous proteins), while ICC/FCM results could be influenced by nonspecific binding or subcellular localization effects.

Figure 2. Validation of CREB1 antibody expression in wild-type (WT) and CREB1 knockdown (KD) HeLa cell lines using Western blot (WB), flow cytometry (FCM), and immunocytochemistry (ICC).

A: Western blot detection of CREB1 protein expression in WT and KD CREB1 HeLa cells.

B: Flow cytometry (FCM) analysis of CREB1 protein status in WT and KD CREB1 HeLa cell populations.

C: Immunocytochemistry (ICC) staining showing the subcellular localization of CREB1 protein in WT and KD CREB1 HeLa cells.

qPCR and WB only reflect mRNA and protein expression levels, respectively, but they overlook two critical dimensions:

(1) Cellular heterogeneity – Uneven knockdown efficiency may result in subpopulations of cells that still express residual target protein. This requires precise quantification by FCM.

(2) Spatial resolution – Changes in subcellular localization may affect protein function and must be evaluated by ICC.

As shown in Figure 3, compared to WT cells, KD RAB9A HeLa cells exhibit a complete loss of the RAB9A band in WB (Figure 3A), a leftward shift of the population signal in FCM (Figure 3B), and no detectable RAB9A signal in ICC (Figure 3C), thereby confirming the antibody’s high specificity.

Figure 3. Validation of RAB9A antibody expression in WT and KD RAB9A HeLa cell lines using WB, FCM, and ICC.

A: WB analysis of RAB9A protein expression in WT and KD RAB9A HeLa cells.

B: FCM analysis of RAB9A protein status in the WT and KD RAB9A HeLa cell population.

C: ICC staining to visualize RAB9A protein localization in WT and KD RAB9A HeLa cells.

Figure 4 shows that in WB analysis, the RABEPK signal completely disappears in KD RABEPK HeLa cells compared to WT cells (Figure 4A), indicating high specificity. However, in FCM analysis, no significant difference in RABEPK signal was detected between WT and KD RABEPK cells (Figure 4B). In ICC analysis, RABEPK in WT cells displayed abnormal localization—nuclear signal instead of the expected membrane localization (Figure 4C), suggesting non-specific binding of the RABEPK antibody in the ICC experiment.

Figure 4. Validation of RABEPK antibody expression in WT and KD RABEPK HeLa cell lines using WB, FCM, and ICC.

A: WB analysis of RABEPK protein expression in WT and KD RABEPK HeLa cells.

B: FCM analysis of RABEPK protein levels in the HeLa cell population of WT and KD RABEPK.

C: ICC staining showing subcellular localization of RABEPK protein in WT and KD RABEPK HeLa cells.

2.2 Multi-dimensional Technical Validation Analysis of KD Experiments on the ShGE™ Platform

From these typical cases, it can be observed that the contradictory results in Figure 2 suggest that WB may yield false negatives or that ICC/FCM may produce false positives, requiring further analysis. The consistent results in Figure 3 demonstrate the effectiveness of the multi-dimensional validation approach. The contradictory results in Figure 4 indicate that WB may have failed to detect issues that are revealed by non-specific binding in ICC/FCM. The findings in Figure 4 clearly illustrate why relying solely on WB is insufficient and highlight the value of ICC/FCM in uncovering non-specificity. These cases emphasize the importance of FCM for population-level quantification and ICC for subcellular localization analysis. Relying on a single technique (such as WB) may lead to misinterpretation, whereas combining multiple techniques—WB, FCM, and ICC—allows for a more comprehensive assessment of antibody specificity and the true expression of protein signals, thereby avoiding erroneous conclusions due to the limitations of any single experimental method.

Based on the multi-dimensional technical validation cases of KD CREB1 (Figure 2), KD RAB9A (Figure 3), and KD RABEPK (Figure 4) cell experiments, several clear insights emerge: the consistency or contradiction of results between techniques holds significant reference value; consistent results (such as RAB9A) substantially enhance data credibility; while contradictory results (such as CREB1 indicating a potential WB false-negative risk, and RABEPK revealing non-specificity in ICC/FCM) highlight the blind spots of relying on a single technique and may point to deeper biological issues (e.g., protein conformational changes, compensatory mechanisms) or technical flaws (e.g., epitope masking, non-specific binding). The disappearance or abnormality of expected localization (such as persistent CREB1 signal or nuclear localization of RABEPK) serves as a key indicator for revealing potential functional abnormalities, non-specific binding, or technical issues. When ICC/FCM shows changes in protein localization without changes in total protein levels, further verification by WB is needed to exclude interference from antibody cross-reactivity. The core advantage of this strategy lies in its complementary dimensions: FCM addresses cellular heterogeneity, ICC analyzes spatial localization, and WB confirms molecular identity. The combination of these three forms a rigorous cross-validation system that significantly improves the comprehensiveness and reliability of antibody specificity assessment and protein signal expression analysis (as demonstrated in the RAB9A case). Its value lies not only in confirming successful KD (e.g., RAB9A) but also in identifying and diagnosing contradictions and anomalies during validation (e.g., CREB1, RABEPK), providing clear directions for subsequent mechanistic research or technical optimization.

3. Summary

In antibody-dependent functional studies and KD efficacy validation, a multi-dimensional cross-validation strategy should be adopted as the standard procedure. The ShGE™ platform verifies antibody specificity by rigorously implementing a stepwise validation process that includes FCM screening, ICC localization analysis, and WB molecular weight confirmation. This approach aims to obtain reliable data, avoid “validation pitfalls,” and deepen the understanding of the relationship and implications between KD target genes, their corresponding protein signals, and highly specific antibody validation. GenuIN Biotech is committed to building a high-quality research reagent system centered around the ShGE™ platform, continuously expanding its KD-validated antibody product library. Meanwhile, the platform has established verified lentiviral vector libraries, stable cell line libraries, and cell lysate libraries, laying a solid foundation for the future development of highly reliable research products and the provision of customized validation services.

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08/14/2025

Xiaohong Lian

Supervisor of Production

GenuIN Biotechnologies