Protein Screening on Simple Western

Introduction

When expressing an antibody or recombinant protein in a heterologous host, getting to the right clone quickly is critical. Obtaining enough material to verify clone positivity can require growth or expansion of the clones, so there is a need for a sensitive detection method to minimize the amount of sample that is required. While sensitive detection of expressed proteins can be achieved by ELISA, it lacks the ability to provide any information on the molecular weight of the protein being detected, making this method insufficient for most clone selection. On the other hand, while the traditional Western blot can provide molecular weight information, it is often not as sensitive as an ELISA and it is even more time consuming and labor intensive.

ProteinSimple’s Simple Western™ offers a powerful solution by coupling protein separation and sensitive detection in high-throughput. This allows you to screen up to 96 samples per day in a single run, and obtain truly quantitative data with molecular weight for the expressed protein. Here, we describe a protocol for Simple Western that will allow you to get the most out of your instruments. In addition, the ability to prepare and freeze your Simple Western samples a day ahead allows for continuous screening throughout the week, up to 480 samples per week per instrument. We’ve also got epitope-tag antibody recommendations so you can start screening with no optimization of the detection. Finally, we describe examples of how you can use this protocol for common tasks, including screening for His tag proteins, epitope tag antibody optimization, and screening for BSA contamination.

Recommended Tag Antibodies

We’ve optimized the most popular HRP-conjugated epitope-tag antibodies (HA, His, and FLAG) so you can quickly start screening your samples. Shown in Table 1 are the details including dilution and incubation time for each antibody. Using HRP-conjugated epitope-tag antibodies allows for a single-step assay to be run. Doing so speeds up the overall assay by eliminating the need for additional detection through a secondary antibody.

To demonstrate the utility of using Simple Western for screening, we’ve included some examples for Jess™, Abby™, or Wes™ and Peggy Sue™ or Sally Sue™

Results

Example 1: His-tagged protein screen on Jess, Abby, or Wes

14 HEK293 clones expressing secreted His-tagged protein constructs and two negative control clones were screened on Jess/Abby/Wes in four consecutive runs on the same day (Figure 4). Each run contained one technical replicate of a given sample and standard curve. Samples for all four runs were prepared in a single 96-well PCR plate the day before the screen (see optional procedure for preparing samples the day before screening). Other reagents (diluted primary antibody and Luminol/Peroxide) where made in bulk, for all 4 Wes runs, and kept on ice for the day protected from light until use.

The His-tagged samples screened on Jess/Abby/Wes resulted in data of high confidence. From Run 1 to Run 4, the average CV was 11.5% for the samples and 10.0% for the standard curve.

Example 2: Multiple epitope tag antibody optimization using a Multi Tag protein

The recombinant E. coli Multi Tag protein from Abcam (ab36791), which contains 12 epitope tags fused in tandem, allows for the optimization of a variety of anti-Tag antibodies with one tool. All it takes is two runs and you’ll have optimized conditions for your anti-Tag antibody of interest. First, titrate both antigen and antibody to get an idea of the affinity of the antibody to the antigen. Then, perform a narrower titration of the antibody with a fixed concentration of the Multi Tag protein (determined in the first experiment). In addition, if you are using a Sue for optimization, you can also conduct a time course to determine the primary antibody incubation time required to reach saturation. This basic approach may also be used for screening multiple antibodies against a given lysate and other samples.

As an example, optimization of the anti-FLAG-HRP (R&D Systems, HAM85291) is shown in Figure 5. The anti-FLAG antibody was titrated against 0.1 mg/mL recombinant Multi Tag protein and shows signal saturation at 1:25 dilution.

Example 3: Screening for protein contamination

Screening on Simple Western is not just limited to finding the right clone; it has also shown great potential in detecting protein contamination in product development that rivals the traditional ELISA-based method of detection. For example, bovine serum albumin (BSA) is a common contaminant in the preparation of vaccines because it is a major component of the culture medium that is used during vaccine production.¹ The World Health Organization guidance requires that no more than 50 ng of BSA be present per human dose of vaccine.² Thus, there is a need for rapidly screening protein contaminants with sufficient sensitivity and reproducibility. Traditional ELISA-based methods are labor-intensive and do not provide size-based separation. Simple Western overcomes these limitations by providing fully automated operation and size-based differentiation, while maintaining the ability to screen large sample sizes (88 samples per run). This means Simple Western not only detects BSA with the necessary sensitivity and reproducibility, but it also distinguishes monomers from oligomers and higher order aggregates. We used the same Peggy Sue protocol as described above to detect BSA in purification fractions by using a rabbit anti-BSA primary antibody available from Bethyl Labs.

Note: Because BSA is present as a blocking agent in Antibody Diluent 2, you should use the Peggy Sue Charge Antibody Diluent (ProteinSimple, 040-309) in place of Antibody Diluent 2 for making antibody dilutions and for blocking.

Briefly, the primary antibody was diluted 1:100 in Charge Antibody Diluent and incubated for 120 minutes, and then Goat-Anti-Rabbit HUX HRP Secondary Antibody (ProteinSimple, 041-081) was diluted to 1X in Charge Antibody Diluent and incubated for 60 minutes. This resulted in a broad range of precise BSA detection (Figure 6).

Conclusion

Simple Western lets you screen large sample sizes quickly while eliminating the manual washing and incubation steps of traditional ELISAs and Western blots. To get you started on your Simple Western with little to no optimization, this present work describes a toolkit for screening some of the most commonly used epitope tags and contaminating proteins. Of course, the Simple Western platform is not limited to the antibodies listed here; any of these antibodies may be swapped out with your antibody of interest to meet your needs. No matter the antibody and target antigen you choose, the straightforward protocol described here will remain essentially the same.