Institutional Development Award
|Contact Information for Nevada Proteomics Center|
|Phone||(775) 784-6337: 2-D Gels|
|Phone||(775) 784-1590: Mass Spectrometry|
|Location||School of Medicine
Manville Medical Building, Rooms 15-17, MS 0330
|Address||1664 N. Virginia Street
Reno, NV 89557-MS 0330
The 2-D gel lab of the Nevada Proteomics Core Facility is a dedicated facility containing all equipment necessary for sample preparation, running of 1-D or 2-D gels (including DIGE gels), staining and imaging of gels, computer analysis and determination of differential expression, as well as excision of spots.
The 2-D gel laboratory contains all equipment and supplies necessary for most sample preparation and clean-up techniques used for bacterial, fungal, plant, insect and mammalian samples.
Samples may be prepared by customers or by the Nevada Proteomics Center. The perfect sample for 2-D gel analysis contains protein but few nucleic acids, salts, complex carbohydrates or lipids. A pellet of precipitated protein or a pellet of washed membranes is usually the ideal sample. Our protocol section contains suggested precipitation and clean-up procedures.
Isoelectric focusing separates proteins by their isoelectric point (the pH at which a protein has no net charge). This is usually done as the first step in 2-D gel electrophoresis; for 2-D electrophoresis, separation is performed on immobilized pH gradient (IPG) strips which vary in length from 7 to 24 cm. Our lab recently acquired a Bio-Rad i12 IEF cell, which independently controls the voltage of each IPG strip, allowing samples of various compositions or strips of various pH ranges to be run simultaneously.
Proteins may also be separated by in-solution isoelectric focusing using our Bio-Rad microRotofor Cell. This method produces fractions which can be analyzed on 1-D gels or by LC/MS. In-solution focusing may provide an excellent separation method for proteins, such as membrane proteins or very large proteins, that are difficult to handle on IPG strips.
The 2-D gel lab can perform both 1-D and 2-D gel electrophoresis using 13.3 x 8.6 cm, 18.3 x 19.3 cm or 25.6 x 23.0 cm gels. We have recently purchased an HPE FlatTop Tower (Gel Company) for horizontal running of plastic-backed gels. Our equipment allows us to run from 1 to 12 gels at the same time. Gels may also be blotted using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) for blotting of 3 Protean Plus, 3 Protean II or 12 Criterion gels.
Gels are routinely stained using Bio-Safe Coomassie (Bio-Rad), Sypro Ruby (Bio-Rad) or Lava Purple (Gel Company). Blots may also be stained before Western blotting with Sypro Ruby blot stain or Lava Purple.
Gels and blots are routinely imaged with our new Bio-Rad ChemiDoc MP which detects colorimetric, fluorescent or chemiluminescent stains. This imager is capable of imaging samples with multiple fluorescent dyes.
Our Typhoon Trio (GE Healthcare) is a very sensitive laser imager used to image DIGE gels or other fluorescent or chemiluminescent gels or blots. It is capable of detecting multiple fluorescent dyes on one gel in a non-overlapping manner.
Quantity One or ImageLab software (Bio-Rad) is used for analysis of 1-D gels. Standard 2-D gels are analyzed using PDQuest v 8.0.1. DeCyder v 7 (GE Healthcare) is very powerful software dedicated solely to analysis of DIGE gels.
Spots of interest are excised from gels using either our ExQuest spot cutter (Bio-Rad) for general cutting of 1-D and 2-D gels or our ProPic II (Digilab) for cutting of DIGE gels
Customers are encouraged to consult with the laboratory staff before initiating proteomics experiments. We are always glad to advise customers as to the most fruitful approaches in order to gain the desired information and to share sample preparation protocols.
DIGE stands for Differential Gel Electrophoresis. It is a powerful method for determining differential expression of proteins in control vs experimental samples. Standard 2-D gels are notorious for poor gel-to-gel reproducibility. When comparing one physiological condition against another, a number of gels must be run for each condition so that changes in protein quantity can be attributed to the physiological change instead of gel-to-gel variability. Because replicate gels display such variability, significant changes of less than 2-fold are seldom found. DIGE, however, overcomes many of these difficulties. In DIGE experiments, three different samples are loaded onto one 2-D gel. Before loading, one sample is labeled with one fluorescent dye (e.g., Cy3), a second sample is labeled with a different fluorescent dye (e.g., Cy5) and a standard that is loaded on all gels in the experiment is labeled with a third dye (e.g., Cy2). After 2-D electrophoresis, gels are imaged with an imager that can detect each of the dyes in a non-overlapping manner. When analyzed with DeCyder software, significant changes of as little as 10% can be seen.
To obtain publishable results, replicate gels, each containing a different biological replicate of the control and experimental samples, should be run. The minimum number of replicate gels for any one experiment is 3. This allows for statistical analysis of the validity of differential protein change seen on the gels.