Analysis of Abnormal Western Blot Results and Their Relationship with Sample Selection and Processing

Western blot, also known as immunoblotting, is a technique used to detect andanalyze the expression of specific target protein signaling molecules in a sample, based on the specific binding reaction between antigens and antibodies. First, proteins with different molecular weights and charges from cells or tissue samples are separated by gel electrophoresis. The separated proteins are then transferred from the gel onto a nitrocellulose (NC) membrane or polyvinylidene fluoride (PVDF) membrane. Subsequently, specific antibodies are used to bind to the corresponding target protein antigens on the membrane. The binding is visualized using chemiluminescence or colorimetric detection. By analyzing the position and intensity of the signal, the expression level of the target protein can be determined.

Western blot (WB) is one of the most commonly used laboratory techniques for detecting protein signaling molecules. The selection and proper preparation of samples are critical for obtaining accurate and reliable results.When WB experiments yield no protein signal (a blank membrane), weak target protein bands, or high background across the entire membrane, researchers often focus on potential issues in experimental procedures or antibody specificity. However, problems related to the characteristics, selection, or handling of the sample and target protein itself are often overlooked.This article provides an analysis of how sample-related factors contribute to common WB abnormalities.

1. Importance of Sample Selection

High-quality samples are the foundation for obtaining reliable results. Degraded or contaminated samples can lead to unclear bands or produce false-positive/false-negative results. Additionally, the selected samples should accurately reflect the research objective.If the chosen cells or tissues do not express the target protein, if the expression level is too low, or if certain proteins require specific stimulation to be detectable, this can result in blank membranes or faint target bands in the WB experiment.

The following examples—UCHL1, CD4, and PARP1—illustrate the importance of selecting appropriate samples and including positive controls in the experiment.

1.1 Target Protein Not Expressed in the Selected Sample

First, it is important to consult databases such as The Human Protein Atlas, UniProt, or relevant scientific literature to verify whether the target protein is expressed in your experimental samples—whether cells or tissues.It is important to note that target protein expression is highly specific to certain cell or tissue types. If the selected sample does not express the target protein, the result of the WB experiment may be a blank membrane. For example, UCHL1 is not expressed in HT-080 cells (Figure 1) or HeLa cells (Figure 2), resulting in no detectable signal for the target protein in the WB experiment (Figure 3).

Figure 1  Expression of UCHL1 in various cell lines: UCHL1 is not expressed in HT-1080 cells.

Figure 2  Expression of UCHL1 in various cell lines: UCHL1 is not expressed in HeLa cells.

Figure 3  Western blot analysis of UCHL1 expression in cells and tissues: No UCHL1 expression was detected in HT-1080 and HeLa cells.

1.2 Low Expression of Target Protein

Databases such as The Human Protein Atlas, UniProt, or relevant scientific literature can also be used to determine the expression levels of target protein in different cells or tissues. If the selected cells or tissues express the target protein at low levels, the resulting WB signal may be very weak or even undetectable. It is important to note that using a sample known to express the target protein as a positive control is an effective way to rule out experimental errors when encountering weak bands.For example, CD4 is expressed at low levels in HT-1080 cells (Figure 4), Ramos cells (Figure 5), and HAP-1 cells (Figure 6), resulting in faint target bands, high background, or even blank membranes in WB experiments (Figure 7).

Figure 4  Expression of CD4 in various cell lines: CD4 is expressed at low levels in HT-1080 cells.

Figure 5. Expression of CD4 in various cell lines: CD4 is expressed at low levels in Ramos cells.

Figure 6. Expression of CD4 in various cell lines: CD4 is expressed at low levels in HAP-1 cells.

Figure 7. Western blot analysis of CD4 expression in cells: CD4 is weakly expressed in HT-1080, Ramos, and HAP-1 cells.

1.3 Inducible Expression of Target Protein

Third, databases such as The Human Protein Atlas, UniProt, or relevant scientific literature can be consulted to determine whether the expression of the target protein requires induction or stimulation.

Some target proteins are not expressed under normal conditions in certain cells or tissues and can only be detected after induction with specific stimulants. If researchers are unfamiliar with the expression characteristics of the target protein in the selected sample, the WB experiment may result in no signal or a blank membrane.

For example, in HeLa and HAP-1 cells, the detection of Cleaved-PARP is dependent on treatment with the inducing agent Staurosporine. In WB experiments, the Cleaved-PARP band is present only after stimulation, but absent without induction (Figure 8).

Figure 8. Western blot analysis of Cleaved-PARP expression in HeLa and HAP-1 cells.

Lanes 1 and 3: Cells without Staurosporine treatment show no detection of Cleaved-PARP.

Lanes 2 and 4: Cells treated with Staurosporine show strong detection of Cleaved-PARP.

2. Sample Processing Issues

The choice of sample directly affects the reliability, reproducibility, and accuracy of Western blot (WB) experiments. However, proper sample handling is also a critical step to ensure that the experimental results are accurately presented.

If there are issues with the processing of cell or tissue samples during WB experiments, the target protein in the sample may exhibit the following problems:

2.1 Low Protein Yield or Degradation

In addition to the previously mentioned reasons—such as low or absent expression of the target protein in selected cells or tissues—low protein content in samples may also result from two common issues:

1. Incomplete cell or tissue lysis: During sample preparation, insufficient cell lysis can prevent the release of most intracellular proteins, and inadequate tissue grinding/homogenization can result in poor protein extraction. This leads to significant protein loss in the final sample.

2. Protein degradation: If the sample is not collected, processed, or stored under cold conditions, or if insufficient protease inhibitors are added, protein degradation can occur, leading to loss of target protein.

As a result, the final WB outcome may show no signal or missing bands for the target protein.

For example, we extracted total protein from HeLa cells using both the IntactProtein™ Universal Protein Extraction Kit (Cat#415) and RIPA buffer. Western blot analysis (30 μg loading) was used to detect mTOR protein expression. The results showed that RIPA buffer failed to fully lyse the cells, resulting in weak protein signals due to incomplete protein release (Figure 9).

Figure 9. Western blot analysis of mTOR protein expression in HeLa cells.

2.2 Incomplete Denaturation

During the processing of cell or tissue samples, if the heating time is insufficient or the temperature is too low, the target protein signaling molecules may not be fully denatured. Incomplete denaturation can affect the electrophoretic mobility of the proteins in the gel, resulting in irregular or blurred bands for the target protein in the final Western blot analysis.

2.3 Contaminants and Non-Specific Binding

During the preparation of cell or tissue samples, if cell debris, nucleic acids, or other impurities are not adequately removed from the lysate, these contaminants can lead to non-specific binding with antibodies. Additionally, if the antibody lacks high specificity, it may bind not only to the target protein but also to other structurally similar proteins in the sample this is known as cross-reactivity. As a result, the Western blot experiment may show non-specific bands, making it difficult to accurately interpret the expression of the target protein.

2.4 High Protein Concentration or Aggregates

During the preparation of cell or tissue samples, if the protein concentration is too high, the migration of proteins through the gel can become inconsistent due to intermolecular interference.Additionally, improper storage conditions or errors during dilution can cause proteins to form aggregates of varying sizes, which also migrate unevenly during electrophoresis.As a result, the Western blot may show smearing or tailing in the target protein bands.As shown in Figure 10, we performed WB to detect AKAP8 expression in HeLa cells. Lane 1 was loaded with 15 μg of total protein, and Lane 2 with 30 μg. The results indicate that excessive protein concentration leads to band tailing.

Figure 10. Western blot analysis of AKAP8 expression in HeLa cells.

3. Solutions from GenuIN Biotech

To avoid abnormal results in Western blot experiments caused by sample-related issues, it is essential to strictly follow standardized protocols during all stages of sample collection, processing, and storage. This ensures the quality and stability of the target protein within the samples. Shanben Biotech recommends that researchers first familiarize themselves with the characteristics of the target proteins in their samples. This can be done by consulting authoritative database resources such as The Human Protein Atlas, UniProt, or relevant scientific literature to verify the expression levels of the target protein. Alternatively, users can refer to the positive control samples provided in GenuIN Biotech’s product catalog. If samples confirmed to express the target protein are used and sample handling is flawless, yet no positive signal or abnormal results are detected, the issue may lie within the WB experimental system itself. GenuIN Biotech has accumulated a comprehensive set of rapid troubleshooting strategies for WB experiments and is ready to provide technical support and services to help users reduce trial-and-error costs. In fact, our company uses an optimized, high-throughput WB system along with our proprietary universal cell/tissue lysis reagent kit for sample preparation daily as part of a larger platform we developed called the ShGE™ gene silencing platform. This platform we use to not only optimize WB, but also to validate the specificity of antibodies by eliminating the target protein—either by gene knockdown (KD) or gene knockout (KO)—and then testing the presence of the antibody signal, which is the globally recognized gold standard of antibody validation. We have already launched over 1,500 highly specific antibodies, lentiviral vectors, and cell lysate products validated by our KD technology .GenuIN Biotech’s technical strengths and products provide reliable resources and services for life science and biomedical basic research, CRO clinical trials, and biopharmaceutical development. We warmly welcome inquiries and collaboration opportunities.

07/30/2025

XiuZhen Cui

Quality Manager

GenuIN Biotechnologies