Table 1.
Table showing the primary antibodies used and basic information about these antibodies.
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.
Table 2.
Western Blotting Minimal Reporting Standard (WBMRS).