An Automated System to Improve Western Blot Processing

Western blot assays1 provide important information about the presence and levels of protein expression. The first step in the procedure is the lysis of cells or tissues followed by solubilization of the protein samples, usually by means of sodium dodecyl sulfate (SDS) and reducing agents. Next, the material is separated by a polyacrylamide gel with or without the denaturing agents and the antigens are transferred to a membrane. The transferred proteins are bound to the surface of the membrane, where they are accessible to the immunodetection reagents. The immunodetection protocol consists of multiple steps including blocking of the nonspecific binding sites, incubation using primary and secondary antibodies and extensive washing between steps. The resulting complex is detected by fluorescence or by using appropriate chromogenic or luminescent substrates, depending on the type of detection agent or enzyme conjugated to the secondary antibodies.

Manual western blotting is a labor-intensive, time-consuming process. The quality of results is dependent on multiple subjective and objective factors such as the qualification and technical skills of the personnel performing the assay and the accuracy of the control of time and temperature, especially during the immunodetection step.

Automated processing

The BlotCycler automated western blot processor (Precision Biosystems, Mansfield, Mass.) was developed to eliminate the variability associated with manual immunodetection procedures and achieve standardized, quality results.

The system (Figure 1) contains several components. Washing buffer is stored in a large tank situated at the top of the system. Twelve smaller vessels inside the tank (six on each side) are used to house and dispense primary and secondary antibodies. The intuitive, easy-to-use touchscreen on the front of the instrument allows the user to program the length of each incubation and wash steps as well as the number of washes after each incubation (Figure 2). The system can also be programmed to skip one or more steps in the protocol.

Figure 1 – BlotCycler automated western blot processor. View from a) side and b) top.
Figure 2 – The intuitive touchscreen menu allows quick and easy protocol customization.

The blot processor is supplied with four preprogrammed protocols, and up to 20 additional custom-written protocols can be added. The device controls the liquid volumes and the sequence of the fluids to be discharged into each individual tray. Blots are processed in trays that can be either 10 cm × 8 cm for mini-gels or 15 cm × 10 cm for midi-gels. Up to two full-size blots can be processed simultaneously per tray. Three tray configurations are available: six mini-gels, four midi-gels and three mini-gels plus two midi-gels. Users can easily switch the tray sets by removing screws located on the instrument’s side panels. Up to two different protocols, six different combinations of primary and secondary antibodies and 12 blots can be run simultaneously.

A patented fluidic control system with automated shaking facilitates consistent and reproducible dispensing, mixing and distribution of the reagents throughout the process. Receptacles for the vials are located at the bottom of the instrument’s side panels to permit collection and recycling of primary antibodies. Trays are designed to ensure efficient mixing of reagents, achieve low background and minimize the amount of antibodies required for analysis. Separate conduits for each primary and secondary antibody prevent potential cross-contamination.

In this study, automatic and manual processing were compared using a fluorescence-based detection system that allowed the simultaneous detection of staining efficiency by up to three different primary antibodies.

Increased sensitivity with automation

Incubation with primary antibody is typically done overnight at 4 °C, with the remaining steps performed at room temperature (RT). (In this way, users do not have to go in and out of the cold room for solution changes.) However, the change in temperature associated with this procedure has been shown to eliminate the advantages of incubation of primary antibody at low temperature on assay background and signal intensity.

To further understand how automation and incubation temperature can result in increased signal, both manual and automatic processing were carried out at 4 °C and RT (Figure 3). The results indicate that, while both automation and a temperature of 4 °C alone increased the signal 1.5- to 2-fold each, the effect was most pronounced when low temperature and automation were combined (up to 4-fold). Therefore, the BlotCycler offers improved western blotting sensitivity while eliminating time-consuming tasks associated with manual processing.

Figure 3 – a) Blots that were processed as indicated in the text. Lane 1: HeLa whole cell lysate, lane 2: recombinant human IL-2, lane 3: mixture of HeLa whole cell lysate and h-IL2. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred from the gel onto the nitrocellulose membrane. Membranes were blocked for 30 minutes at RT or for 90 minutes at 4 °C using MB-070 blocking reagent (Rockland Immunochemicals, Limerick, Pa.) and then incubated with rabbit anti-IL2 and mouse anti-α-tubulin primary antibodies for 90 minutes at RT or 18 hours at 4 °C, followed by incubation with DyLight549 conjugated anti-mouse IgG and DyLight 649 conjugated anti-rabbit IgG secondary antibodies (Rockland Immunochemicals) for 30 minutes at RT or 90 min at 4 °C. Data for each DyLight fluorophore was collected independently at the following excitation/emission wavelengths: 530/605 nm for DyLight 549 and 625/695 nm for DyLight 649. b) The fluorescent intensity between different blots was normalized using MW standard.

Reproducibility test

To determine if the automated western blot processing results in a reproducible signal, five identical blots were processed using the same antibodies described in Figure 2. The results in Figure 4a demonstrate excellent reproducibility. To confirm this quantitatively, the band intensity for each protein was quantified and normalized against the marker. The calculations shown in Figure 4b are consistent with the observations and suggest that the salient characteristics of BlotCycler, 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.

Figure 4 – a) Five identical blots. Lane 1: HeLa whole cell lysate, lane 2: recombinant Human IL-2, lane 3: mixture of HeLa whole cell lysate and h-IL2. All incubation steps were performed at 4 °C using the antibodies and protocol described in Figure 3. b) The calculated fluorescent intensity between different blots was normalized using MW standard.

Throughput, reproducibility and rigorous optimization of antibody concentration

Optimal concentrations of both primary and secondary antibodies against a given antigen are essential to obtain the most informative results. BlotCycler allows simultaneous processing of up to six different blots with six secondary or primary antibodies, thereby avoiding the inherent variations associated with the manual processing of multiple blots (see Figure 5). Ten identical strips that contained a molecular weight (MW) marker and mouse pancreas extract were prepared and probed with five different dilutions of primary antibody against Delta-4 (Figure 5a) and a single dilution of secondary antibody. The second experiment contained a single dilution of primary antibody and six different dilutions of the secondary antibody (Figure 5b). The titration curves for both the primary and secondary antibodies were constructed using the blot processor.

Figure 5 – 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 (1:1000). The gel was loaded with an extract of mouse pancreas in all lanes. All incubation steps were performed at 4 °C following the same protocol as described in Figures 3 and 4.

Summary

The BlotCycler automated western blot processor eliminates the need to watch over the procedure, which is necessary with manual immunodetection. The system provides reproducible, quality staining, recycling of primary antibodies and optimization of their concentrations, which results in cost savings and increased productivity.

Reference

  1. Towbin, H.; Staehelin, T.; et al. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. Sept 1979, 76(9), 4350–54.

Dr. Margulis is an application scientist, Precision Biosystems, LLC, 241 Francis Ave., Mansfield, Mass. 02048, U.S.A.; tel.: 888-490-4443, ext. 1; e-mail: [email protected]; www.precisionbiosystems.com. Ms. Zaorski is a scientist, Brentwood Industries, Inc., Reading, Pa., U.S.A. Dr. Chimento is an assistant laboratory director, Rockland Immunchemicals, Inc., Limerick, Penn., U.S.A. Dr. Yukhananov is CEO, Precision Biosystems.

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