Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Abstract

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

Fig 1. Comparison of anti-ubiquitin antibodies.

Heart and liver lysates (20 μg each) were investigated by Western blotting using five commercially available anti-ubiquitin antibodies (VU101, U5379, AP1228a, P4G7-H11, FK1). Arrow shows location of free unbound ubiquitin. Stain-free staining of total proteins loaded was used as the normalization control. H, heart; L, liver. BSA was used as the blocking reagent for the blot labeled FK1* while non-fat milk was used as the blocking reagent in all the other blots shown. All antibodies were used at a dilution of 1:1000 except for blots labeled U5379* and U5379^ which were used at dilutions of 1:100 and 1:2000 respectively.

Fig 2. Validation of anti-ubiquitin antibodies.

VU101 in the presence and absence of 0.5% glutaraldehyde pre-treatment, U5379, AP1228a, or P4G7-H11 were used to detect ubiquitin and ubiquitinated proteins. A) Western blot of polyubiquitin chains (Ub3, Ub5, Ub8) (lane A), purified ubiquitin (lane B), polyubiquitinated proteins from H9c2 cells treated with 10μM MG-132 for 36 h obtained from affinity purification using TUBEs (lane C), and unbound fraction from H9c2 cells after removal of polyubiquitinated proteins (lane D). B) Upper figure, Western blot of free ubiquitin (lane A) and polyubiquitin chains (lane B) with U5379 antibody diluted at 1:100 and 1:2000. Lower figure, Western blot of free ubiquitin (lane A) and polyubiquitin chains (lane B) with FK1 antibody diluted at 1:1000 in BSA. Even when the blots were imaged for long time periods no additional bands were seen.

Fig 3. Comparison of anti-ISG15 antibodies.

(A) Seven anti-ISG-15 antibodies were used to detect the levels of ISGylated proteins in four different types of samples. (B) Quantification of ISG15 Western blots. Young, 10 month old hearts; Young HLS, high-limb suspended 10 month old hearts; old, 30 month old hearts; Old HLS, high-limb suspended 30 month old hearts. * p < 0.05, ** p < 0.01 by 1-way ANOVA.

Fig 4. Effect of antibody concentration on linearity of target proteins detected by Western blotting.

(A) Western blot of rat liver samples (3–12 μg) using anti-PSMA6 at four different concentrations (1:5000, 1:10000, 1:20000, and 1:50000). (B) Quantification of anti-PSMA6 Western blots without including any normalization. (C) Quantification of anti-PSMA6 Western blots using total protein normalization. (D) Western blot of rat liver samples (3–12μg) using anti-β-actin at four different concentrations (1:1000, 1:2500, 1:10000, and 1:25000). (E) Quantification of anti-β-actin Western blots without including any normalization. (F) Quantification of anti-β-actin Western blots using total protein normalization. * p < 0.05 by 1-way ANOVA.

Fig 5. Effect of buffer reagent on Western blotting linearity.

(A) Western blot of rat liver samples (3–12 μg) using anti-PSMA6 and different buffers (TBST and PBST). (B) PSMA6 quantification, not normalized to total protein. (C) PSMA6 quantification, normalized to total protein. (D) Western blot of rat liver samples (3–12 μg) using anti-β-actin and different buffers. (E) β-actin quantification, not normalized to total protein. (F) β-actin quantification, normalized to total protein. * p < 0.05, ** p < 0.01 by 1-way ANOVA.