We provide 1D and 2D gel services. We encourage and willingly provide support to internal users to run and process gels in the facility.
- 1D-SDS-PAGE proteins are separated according to molecular mass. This is good for simple samples and hydrophobic proteins.
- 2D-Gels Here, the initial protein separation is done through isoelectric focusing using the BioRad Protean IEF Cell or IPGphor from Amersham Biosciences. Proteins are then separated by molecular mass using Dodeca Cell from BioRad or Ettan Daltsix system from Amersham Biosciences. This is a traditional 2D PAGE, which can resolve a large numbers of proteins (up to 2000) on a single gel. When stained with dyes of high sensitivity and dynamic range, protein expression levels can be quantified, thus enabling global protein expression analysis.
Imaging and Image Analysis
Proteins in gels must be stained or labeled
in order to visualize the proteins. The facility uses several stains, the choice of which depends on the downstream mass spectrometric analysis.
- Colloidal Coomassie Blue (Bio-Safe Coomassie): it stains the broadest spectrum of proteins. It has about 2 orders of magnitude and sensitivity down to 10 ng.
- SYPRO Ruby fluorescent staining: it gives little background staining and is very sensitive (1 ng). The stain is linear over 3 orders of magnitude which is very useful for quantitative analysis. The excitation peaks of the gel stain are at 280 nm and 450 nm, and the emission maxima is near 610 nm.
- Silver Staining: it is very sensitive (1 ng), but the linearity is low (1 order magnitude). Be aware that traditional silver staining involving oxidization of proteins is not compatible with mass spectroscopic analysis since the oxidative step changes protein mass. As a result, mass spectrometric compatible silver staining procedures, such as SilverQuest from Invitrogen or Silver Stain Plus from BioRad, should be used.
- ProQ Emerald glycoprotein stain reacts with periodate-oxidized carbohydrate groups, creating a bright green-fluorescent signal on glycoproteins. It is possible to detect as little as 0.5 ng of glycoprotein per band, depending upon the nature and the degree of glycosylation. The stain can be visualized with 300 nm UV illumination. After imaging, the proteins can be counter stained with SYPRO ruby to detect all the proteins.
- ProQ Diamond stain allows direct, in gel detection of phosphate groups attached to tyrosine, serine, or threonine residues. The stain allows detection of as little as 1-16 ng of phosphoprotein per band, depending on the phosphorylation state of the protein. ProQ Diamond has excitation/emission maxima of 555/580 nm. After imaging, the proteins can be counter stained with SYPRO ruby to detect all the proteins.
Images from gels stained with Coomassie and Silver can be acquired with a flatbed scanner (Epson Expression 1640XL). For Sypro and ProQ Diamond stained gels, a Typhoon 9410 imager is used. The ProQ Emerald stained gels can be imaged using a UV transilluminator.
After image acquisition of replicate gels of different samples, the images need to be analyzed using computer-assisted 2D gel image analysis software (Progenesis SameSpot) which does spot detection, alignment, matching, spot normalization and quantitation, pI and MW determination, gel average and comparison, statistical analysis, annotation and documentation.