Fig. 1. Workflow of a typical immunodetection protocol by western blot. After nonspecific binding sites are blocked, usually by adding dry milk or Bovine Serum Albumin (BSA) to a wash buffer saline solution that contains a mild detergent, the membrane is probed with the primary antibody and then washed 3-5 times. The antibody-antigen complexes are then detected by a secondary antibody conjugated to a fluorophore (A) or an enzyme such as Horseradish Peroxidase (HRP) that converts a substrate into a light-emitting molecule (B). After 3-5 washes, the resulting complex is detected by fluorescence or chemiluminescence respectively.

Fig. 2. Automated western blot processing by BlotCycler increased the signal as compared to the manual procedure. (LEFT) Images of the blots processed as indicated. In each blot, the first lane contained a HeLa whole cell lysate, the second lane contained a recombinant Human IL-2, and the third lane a mixture of HeLa whole cell lysate and h-IL2. Following the SDS-PAGE electrophoresis, proteins were transferred from the gel onto the Nitrocellulose membrane. Membranes were blocked for 30 min at RT or for 90 min at 4ºC using MB-070. Rabbit anti-IL2 and mouse anti-α-tubulin primary antibodies were incubated for 90 min at RT or for 18 hours at 4°C. DyLight™ 549 conjugated anti-mouse IgG and DyLight™ 649 conjugated anti-rabbit IgG secondary antibodies were incubated for 30 min at RT or 90 min at 4°C. Data for each DyLight™ fluorophore were collected independently at excitation/emission wavelengths: 530nm/605nm for the DyLight™ 549 and 625nm/695nm for DyLight™ 649. The fluorescent intensity between different blots was normalized using MW standard as shown in (RIGHT).

Fig. 3. Automatic western blot processing by BlotCycler yielded highly reproducible results. (TOP) 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 (BOTTOM).

Fig. 4. BlotCycler enabled optimization of antibody concentrations. (A) Titration of the primary rabbit anti-Delta-4 antibody with a fixed secondary DyLight™ 649 conjugated anti-rabbit IgG antibody concentration (1:20,000). (B) Titration of the secondary antibody with a fixed primary antibody concentration (1:1,000). The gel was loaded with an extract of mouse pancreas in all lanes. Electrophoresis was run as described. Proteins were transferred from the gel onto the Nitrocellulose membrane. Membranes were blocked for 90 min using MB-070 and then incubated with the primary antibody for 18 hours, followed by incubation with the secondary antibody for 90 min. All incubation steps were performed at 4°C. Data was collected independently using a bandpass filter at 625/695 nm for DyLight™ 649. The fluorescent intensity between different blots was normalized using MW standard.