SAMPLE PREPARATION

Sample preparation is the most critical step in obtaining good results from 2DE. Samples can be obtained from tissues or cultured cells.

The quality of the gel depends on the types and amounts of detergents, salts, contaminants, and other interfering material present in the sample.

SAMPLE LABELING

Samples are labeled with specific fluorescent dyes. Up to three different protein samples can be labeled for 2DE.

The spectral separation allows for detection of real differences in protein expression.

The major source of error in 2DE is gel-to-gel variation. This is eliminated by loading different samples in the same gel.

SAMPLE FRACTIONATION BY 2DE

Individual proteins from each sample set are isolated by electrophoresis.

The first dimension consists on the separation of proteins in a mixture based on their individual isoelectric points, using an Isoelectric Focusing apparatus.

For the second dimension proteins are separated in an acrylamide gel.

The fractionation property is the individual molecular weight of each protein.

The second dimension apparatus shown in the picture is designed to run up to six gels simultaneously, in a 24x24 cm format. Other formats are also available in the NPL.

GEL IMAGING

Each individual set of proteins is visualized in the mixture based on the spectral properties of the labeling dyes.

The imager is able to separate the spectra of images that look as single spectrum with other types of visualizations.

The Typhoon 9400, shown at left, uses three laser beams. The excitation energy emitted for each laser is specific for a single dye, allowing for the digital separation of the images from a complex mixture.

IMAGE ANALYSIS

Individual spots (proteins) are analyzed as peaks characterized by their intensity and shape with the DeCyder software.

Spots are analyzed with a powerful software that identifies differential expression of proteins (performed with the Differential in Gel Analysis component, DIA). The correlation between spots is used to identify Down or Up regulation and biological relevance as compared with a control sample (The Biological Validation Analysis is carried out with the BVA component of the DeCyder software).

SPOT PICKING

Spots that are identified as having biological relevance are cut with the Ettan spot picker.

Spots (proteins) in the gel are transferred to 96 well plates for digestion. Several digestive enzymes can be used to generate corresponding peptides that will be used to identify the proteins by Mass Spectroscopy.

The spot picker is designed to cut and transfer the gel fragments to well plates with a 99.98% accuracy.

Protein digestion and identification is performed in the Mass Spec facility, Department of Biochemistry.