Services: Protein Profiling – 2-D DIGE analysis and sample requirements

Protein profiling determines the molar ratios (relative abundance) of identical proteins in compared complex protein mixtures (protein samples). 2-Dimensional Fluorescence Difference Gel Electrophoresis- 2-D DIGE enables protein profiling in multiple multi-protein extracts. This is achieved by labeling of each multi-protein extract (a sample) using spectrally resolvable, size and charge-matched fluorescent dyes known as Cy2, Cy3, and Cy5 fluorophores. Equal amounts (50 ug) of three protein extracts labeled with three different fluorescent dyes are mixed and proteins are separated by two-dimensional (IEF followed by SDS-PAGE) gel electrophoresis. Identical proteins originating from different samples and labeled with different fluorophores are resolved in a single spot on a 2D gel. If losses occur during analysis, each sample experiences the same loss and the ratios of identical proteins are preserved. Protein spots on a 2D gel are detected with high sensitivity by detecting fluorescent labels (2D gel image). All proteins separated on a 2D gel are quantified based on the corresponding fluorescence intensities and the relative abundance (molar ratio) of identical proteins within each spot is determined. Though, up to three samples can only be analyzed at a time (per gel), the method allows protein profiling in higher number of samples by involving a reference sample and multiple gels. A reference sample consists of an equimolar pool of all or several samples, where all proteins of the compared samples are present and the content of each protein possibly varying from sample to sample is averaged. The reference sample is labeled with Cy2 and two individual samples are labeled with Cy3 and Cy5. The reference sample is included on each gel with one or two individual samples (a control or/and a test sample, for example). The abundance of a sample protein relative to the corresponding reference protein – the sample/reference protein ratio is determined in each protein spot on each 2D gel. The presence of the same reference in every 2D gel provides a link between multiple samples analyzed on multiple gels. Based on sample/reference ratios, sample/sample ratios of the corresponding proteins can be determined for each protein separated by 2D gel electrophoresis: the values determined for a given protein in the samples of the same experimental group (control or treated, for example) are averaged and the average values determined for different experimental groups are compared. The ratio of two averages reflecting the relative abundance of a given protein in compared experimental groups is determined and the statistical significance of the possible difference is estimated.

Selected 2D gel spots are analyzed for protein (protein of interest) identification. The selected spots are excised from a preparative 2D gel using an Ettan Spot Picker (GE Healthcare, Piscataway, NJ). Proteins in the excised gel pieces are digested using an in-gel trypsin digestion kit (Pierce, Rockford, IL), and generated tryptic digests are analyzed by LC/MS/MS.

The following services are provided for 2-D DIGE analysis:

Service Service Code Sample Requirements
Protein profiling in two samples (Cy3/Cy5 labeling) per 2-D gel
  • Total protein in a sample: 50-100 ug
  • Total protein concentration in a sample: 2 - 5 mg/ml
  • Sample buffer: 30 mM Tris-HCl, pH 8.5, 7M urea, 2M thiourea, 2% (w/v) surfactant ASB-14
Protein profiling in three samples (Cy2/Cy3/Cy5 labeling) per 2-D gel
Matching and comparative analysis of multiple 2-D gel images with the
same reference (Cy2/Cy3/Cy5 labeling)
Preparative 2-D gel electrophoresis for spot picking - including Sypro Ruby staining, gel imaging, image match to the master image and gel spot picking
Protein identification in picked gel spots through in-gel trypsin digestion and LC/MS/MS analysis