Protein electrophoresis is a separation technique that sorts proteins based on their mobility through a gel. An electric current is applied through the gel, forcing the charged protein molecules to be drawn through the pores of the gel. Separation occurs because some protein molecules move more easily through the gel due to their size and the size of the pores.
Protein electrophoresis services provided by the Proteomics Core include:
- Mini- and midi-sized precast SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels
- 2-D gels in small or large format
- Various staining protocols, including colloidal Coomassie, silver stain, pre- or post-electrophoresis fluorescent labeling, and glycoprotein- or phosphoprotein-specific
- Electrotransfer of mini- or midi-sized gels for subsequent Western blotting
The core can also handle sample preparation, including sample cleanup, protein precipitation, desalting and protein assay.
Additionally, Bio-Rad Quantity One and ProteinSimple AlphaView densitometry software for quantitative gel and Western blot analysis, as well as Bio-Rad PDQuest and Ludesi Redfin programs for 2-D gel analysis, are available for client use. Analysis can also be contracted to Proteomics Core personnel.
SDS-PAGE. Precast Bio-Rad or Invitrogen Novex gels (mini- or midi-size) are used for the separation of proteins based on molecular mass. Up to 12 gels can be run simultaneously. Protein stains available include visible (such as colloidal Coomassie or mass spectrometry-compatible silver stain) or fluorescent dyes; usage depends on sample load, sensitivity, dynamic range and investigator preferences.
Gel images are acquired with a Bio-Rad GS-800 calibrated densitometer, Bio-Rad Molecular Imager FX (scanning wavelengths are 488, 532 and 635 nanometers) or ProteinSimple FluorChem M camera system.
2-D gel electrophoresis. Proteins are separated first on the basis of intrinsic charge (isoelectric focusing, or IEF) and second by molecular mass (SDS-PAGE). Samples can be pre-labeled with fluorescent dyes, such as GE Life Sciences' CyDye DIGE fluors, which are especially useful for matching the complex protein pattern on immunoblots with specific Western spot signals. The same size gels used for 1-D SDS-PAGE are suited for 2-D gels.
Large-format 2-D gels are also a valuable tool for differential protein expression among sample groups using biological replicates. Ideally, projects involving 2-D gels are discussed with Proteomics Core personnel before sample collection and processing, as sample preparation is critical to successful IEF, resolution and separation of proteins. Also, statistical parameters, such as sample size, need to be addressed.
Biological fluids such as serum, plasma and urine pose significant obstacles to differential expression experiments such as biomarker discovery, and special sample preparation parameters such as immunodepletion of abundant proteins are recommended.
- Western blotting. Blotting of gels to polyvinylidene fluoride (PVDF) or other membranes can be done by core personnel using a fast semi-dry method — iBlot by Invitrogen, which takes just seven minutes — or the traditional full-plate tank method. For detection, the FluorChem M can capture chemiluminescent signals, as well as signals from fluorescent-labeled secondary antibody. Western blotting projects can be accommodated on a limited basis.
Project costs are on a per-gel basis or based on instrument time. For sample preparation and analysis, costs are on an hourly basis for technician time.
For more information or to initiate a service or project request, email the Proteomics Core.