Western Blot Processing is Highly Reproducible using the Blotcycler

Blotcycler^TM system’s western blot processing is highly reproducible

To determine if the automated western blot processing results in highly reproducible signal, five identical blots were subjected to the analysis by BlotCycler^TM using the same antibodies as described in Fig. 2. The results on Fig. 3 demonstrated excellent reproducibility as seen visually (Fig. 3A). To confirm this quantitatively, the band intensity for each protein was quantified and normalized against the marker. The calculations shown in Fig. 3B are consistent with visual observations and suggest that the salient characteristics of BlotCycler^TM, such as timing and consistency of solution changes, efficient washing, and elimination of operator caused errors, would enable the much-needed standardization of western blotting.

Fig. 3A                                                                                                             Fig. 3B

Fig. 3. Automatic western blot processing by BlotCycler^TM yielded highly reproducible results.  (A) Images of the five identical blots where the first lane contained a HeLa whole cell lysate, the second lane contained a recombinant Human IL-2, and the third lane had a mixture of HeLa whole cell lysate and h-IL2. After SDS-PAGE electrophoresis, proteins were transferred from the gel onto the Nitrocellulose membrane. Membranes were blocked for 90 min using MB-070. and then incubated with the rabbit anti-IL2 and mouse anti-α-tubulin primary antibodies for 18 hours, followed by the incubation with DyLight™ 549 conjugated anti-mouse IgG and DyLight™ 649 conjugated anti-rabbit IgG secondary antibodies for 90 min. All incubation steps were performed at 4⁰C. Data for each DyLight™ fluorophore was collected independently at excitation/emission wavelengths: 530/605 nm for the DyLight™ 549 and 625/695 nm for DyLight™ 649. The calculated fluorescent intensity between different blots was normalized using MW standard as shown in (B).