>> Update My Profile | Subscribe to Access Agilent | Article Directory

Agilent 2100 Bioanalyzer:
Huge gains in sensitivity for target protein analysis

By Andreas Rüfer
Agilent Product Manager, 2100 Bioanalyzer & 3100 OFFGEL Electrophoresis Systems

	Protein sample (10 µL, 10 µg/µL) Labeling with the HSP250 fluorescent dye (45 min) Preclearing with 1 µL Protein A magnetic beads (10 min) Incubation with specific antibody (1 h) Incubation with 1 µL Protein A magnetic beads (10 min) Wash beads 3x with 200 µL buffer each (15 min) Elution with water + 50% sample buffer with DTT (5 min 95 °C) On-chip analysis with the HSP250 (30 min) HSP250 =  Agilent High Sensitivity Protein 250 assay DTT = dithiothreitol
Figure 1. You can complete this workflow for the specific detection of tagged proteins with the IP/HSP250 method in three hours, versus one day for Western blotting.

Immunoaffinity-based methods like Western blotting and enzyme-linked immunosorbent assay (ELISA) are popular tools for the targeted analysis of proteins in complex samples. Recently, Agilent researchers developed a new, alternative method that combines the specificity of immunoprecipitation with the extreme sensitivity of protein detection on microchips. This IP/HSP250 method is based on the Agilent High Sensitivity Protein 250 kit for the Agilent 2100 Bioanalyzer, and produces faster, more reliable results than Western blotting.

Figure 1 shows an overview of the protocol for the IP/HSP250 method. Steps that use components of the High Sensitivity Protein 250 kit for sample preparation and final analysis are highlighted in yellow.

Increase sensitivity by 1,000-fold

Figure 2. These overlaid electropherograms show that the Agilent IP/HSP250 method allows detection of the target protein at levels down to just 0.001%. (Click here to see this image larger.)

Figure 3. The Western blot method could detect the target protein only at the 1% level. The Agilent method was 1,000 times more sensitive. (Click here to see this image larger.)

The performance of the IP/HSP250 method was initially demonstrated with samples of E. coli cell lysate spiked with a glutathione S-transferase (GST)-tagged protein. Tag-specific antibodies and Protein-A magnetic beads were used to isolate the target protein. Figure 2 shows the results of the IP/HSP250 method from E. coli samples spiked with varying amounts of GST-tagged phosphatase and tensin homolog (PTEN). After subtracting the negative control run, the limit of detection (LOD) was 0.001%, or 100 pg/µL PTEN in a background of 10 µg/µL E. coli lysate.

To benchmark the new method against an established technique for targeted protein detection, Agilent scientists prepared Western blots with the same samples and antibodies. The Western blot with chromogenic immunocomplex detection showed a high non-specific background in all lanes, with a distinct band for PTEN only at the 1% level (Figure 3, left). The gel-like view from the High Sensitivity Protein 250 assay (Figure 3, right) showed the PTEN at much lower levels. The IP/HSP250 method exhibited both higher sensitivity and specificity than Western blot, resulting in a 1,000-fold better limit of detection of the target protein.

Save on time and reagents

Other advantages of the new IP/HSP250 method in comparison with Western blotting are:

• Better accuracy and precision – fewer manual steps and direct availability of quantitative data from the 2100 Bioanalyzer
• Increased productivity – three hours versus one day analysis time
• Reduced cost for antibodies – 10-fold less primary antibody and no secondary antibody needed
• Lower reagent consumption – environmentally friendly process

Compared with conventional Western blotting with chromogenic immunocomplex detection, the new IP/HSP250 method for the targeted detection of proteins delivers superior results for specificity, sensitivity, usability, and time-to-result. If you use immunoaffinity-based methods in studies of protein expression and purification, you may download the corresponding Application Note (5990-4097EN) that provides all the details for these experiments. Then contact your Agilent Representative to learn more about the 2100 Bioanalyzer.

>> Update My Profile | Subscribe to Access Agilent | Article Directory