A new method for the targeted analysis of proteins is presented, which combines the specificity of immunoprecipitation with the sensitivity of protein detection on microchips using the High Sensitivity Protein 250 assay for the Agilent 2100 Bioanalyzer. As as an alternative to Western blotting, this method is valuable for researchers engaged, for example, in protein expression and purification in pharma, biotech, or academic labs. Advantages of this new method in comparison to Western blotting are:

• More reliable results: higher specificity and sensitivity

• Better accuracy and precision: less manual steps and direct availability of quantitative data

• Increased productivity: 3 hours versus 1 day analysis time

• Lower spending for antibodies: 10× less primary and no secondary antibody are needed

• Lower reagent consumption: environmentally friendly process

Introduction

Today, immunoaffinity is a crucial tool for the targeted analysis of proteins in complex samples. Techniques like enzyme-linked immunosorbent assay (ELISA) and Western blotting are widely used for a broad range of applications such as biomarker candidate verification in body fluids or clone selection for recombinant protein expression. The Agilent 2100 Bioanalyzer is a versatile analytical tool that allows characterization of diverse biological samples such as DNA, RNA, proteins, and cells (1). However, most current applications measure all analytes in a given size range (e.g., the Protein 80 assay that determines the size and quantity of all sample proteins from 5 to 80 kDa).

This Application Forum describes an application for the targeted analysis of proteins with the Agilent 2100 Bioanalyzer based on the High Sensitivity Protein 250 assay in combination with immunoprecipitation (IP/HSP250 method; Figure 1). The High Sensitivity Protein 250 assay achieves protein sizing and quantification with sensitivity superior to silverstained SDS-PAGE and a linear dynamic range spanning up to four orders of magnitude (2). Figure 1 shows the workflow of the IP/HSP250 method.

Figure 1. Workflow for the specific detection of tagged proteins with the IP/ HSP250 method. (Click to enlarge)

Highlighted in yellow are steps of the High Sensitivity Protein 250 assay for sample preparation and final analysis on chip (3). Reagents, microchips, and protocols necessary for these steps are supplied with the High Sensitivity Protein 250 kit. The immunoprecipitation was done after sample preparation employing target-specific antibodies and Protein A–coated magnetic beads. Elution of immunocomplexes from the beads was done by heat denaturation in an SDS- and DTT-containing buffer supplied with the High Sensitivity Protein 250 kit. The eluted proteins were directly loaded onto microchips and analyzed automatically with the Agilent 2100 Bioanalyzer. Total time demand for the IP/HSP250 method was ~3 h. Conventional immunoprecipitation techniques that use SDS-PAGE with silver or Coomassie staining for protein detection may have a problematic background, because molecular tools, such as antibodies and capture reagents, are stained as well. This is not an issue with the IP/HSP250 method, since the sample proteins are labeled with a fluorescent dye in the initial sample preparation step. Only these labeled species are detected after the immunoprecipitation by laser-induced fluorescence detection on-chip. The performance of the IP/HSP250 method is shown with Escherichia coli cell lysate samples spiked with a GST-tagged protein and tag-specific antibodies. Western blots were prepared with the same samples and antibodies to benchmark the new method against an established technique for targeted protein detection.

Experimental

Material

Materials needed are Dynabeads Protein A, DynaMag-2, XCell II Mini-Cell & Blot Module, precast 4–12% BisTris minigels, prestained protein standard, PVDF membrane, buffers for SDS-PAGE and Western transfer, WesternBreeze Chromogenic Western Blot Detection Kit (Invitrogen, Carlsbad, CA, USA), Protein LoBind tubes (Eppendorf GmbH, Hamburg, Germany), Zeba Desalt Spin Columns and Coomassie Plus Assay Reagent (Pierce, Rockford, IL, USA), lyophilized E. coli strain B cells (ATCC no. 11303), rabbit polyclonal anti-GST antibody (Sigma-Aldrich, Taufkirchen, Germany), GST-tagged phosphatase and tensin homolog (PTEN), and Agilent 2100 Bioanalyzer and High Sensitivity Protein 250 kit (Agilent Technologies GmbH, Waldbronn, Germany).

Sample preparation

GST-tagged PTEN was supplied as 1 mg/mL solution in 50 mM Tris/HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, and 5 mM DTT. Lyophilized E. coli cells were resuspended in 50 mM sodium phosphate buffer pH 8, 300 mM NaCl, and disrupted with a BeadBeater (Biospec Products, Bartlesville, OK, USA). Cell debris was removed by centrifugation at 12,000× g for 10 min. A buffer exchange to 50 mM sodium phosphate buffer pH 8.3, 300 mM NaCl was done with 2 mL Zeba Desalt Spin Columns. Total protein concentration of the cell lysate was determined with the Coomassie Plus Assay Reagent.

E. coli extract at a concentration of 10 µg/µL was spiked with 1% GST-tagged PTEN (corresponding to 100 ng/ µL target protein). Serial dilutions of this 1% mixture with E. coli lysate were prepared to obtain mixtures with 0.1% (10 ng/µL), 0.01% (1 ng/µL), 0.001% (100 pg/µL), and 0.0001% (10 pg/µL) tagged protein. Control samples of target protein (without E. coli lysate) were diluted with labeling buffer (30 mM Tris/HCl, pH 8.5) to 100 ng/µL.