Protein Picking Season On Automatic

Logic can be quite useful in figuring out magical acts: When dozens of clowns pile out of a tiny car, you can assume that the car is parked over a secret hatch in the ground below. But similar logic doesn’t always apply to the genetic world. How do the genome’s 30,000 genes encode for an estimated 400,000 proteins? Life scientists are now trying to answer that mystery with a number of strategies, many of which include two-dimensional gel electrophoresis (2DGE). Coupled with automated spot pickers, you can incorporate the technique—which has long been central to studying a complex mixture of proteins—with high throughput methods.

2DGE separates proteins based on their isolectric point and molecular weight, which both dictate how individual proteins migrate through the gel. The resulting gel can contain hundreds or thousands of proteins throughout. Staining allows visualization of these protein spots and estimation of relative amounts. To further analyze individual proteins, such as with mass spectrometry, the spot must be excised from the gel. Increasingly, researchers are addressing the irreproducibility and limited sensitivity of 2DGE by employing difference gel electrophoresis (DIGE). The technique relies on using different colored dyes and scanning them with lasers of two distinct wavelengths, producing two gels that can be compared: “Relative increases and decreases in the levels of the proteins can therefore be quantified precisely”.¹

Traditionally, the entire process required several days of manual labor. Automated spot pickers were designed to free researchers from the tedium of hand-picking protein spots. All you need to do is input your desired parameters and let the instrument complete the task, sometimes overnight or over the course of a few days. Once started, the automated picker can record a digital image of the gel, analyze the protein spots, and pick the appropriate ones. Automation not only frees time, but also eliminates human error, which improves accuracy.

The accuracy largely stems from software that follows precise rules and algorithms in analyzing spots. While a good number require data based on DIGE, others can be used with one-dimensional and two-dimensional gels. The analysis helps to determine which spots should be picked. If you’d like to be more involved, consider purchasing instruments that enable you to view a digital image and choose which ones to pick by a mouse click. Advances of imaging technology with higher resolution produce more detailed images, which has improved sensitivity.

Spot pickers come with several convenient features. Most are designed to deposit protein spots into 96-well plates. Look for an instrument that can accommodate the size of your gels. Or, purchase one that is flexible enough for all sizes and formats, such as gels with different backings or none at all. For those fragile proteins, you should consider spot pickers that are enclosed for the control of temperature, humidity, and other environmental factors.

¹MW Duncan and SW Hunsucker, “Proteomics as a Tool for Clinically Relevant Biomarker Discovery and Validation”, Experimental Biology and Medicine, 230(11):808-817, December 2005.