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Sample Preparation & Gel Electrophoresis Services

The sample preparation and gel electrophoresis lab of the Nevada Proteomics 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.

What Services Does the Nevada Proteomics Core Facility Offer?

Sample Preparation

Clean, careful, targeted sample preparation is essential for obtaining valuable information from mass spectrometry experiments. A well-designed sample preparation protocol must avoid contamination by keratin, must extract all proteins of interest and should eliminate proteins that are not of interest. The sample preparation laboratory contains all equipment,supplies and expertise 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 mass spectrometry or 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.

Prefractionation for MS Analysis

For a very complex sample, prefractionation of the proteins in the sample, followed by LC-MS analysis of each fraction, allows for identification of many more proteins than can be seen without such prefractionation. This is usually performed by separating proteins or protease fragments on the basis of charge. The Nevada Proteomics Center has a number of methods for accomplishing such separation.

Proteins or peptides can be separated by strong cation exchange on our new Thermo Scientific Ulti-Mate 3000 HPLC. Fractions are automatically collected by this instrument and then analyzed.

An alternative method for separating by charge is in-solution isoelectric focusing. The Sample Preparation lab has two different instruments that can accomplish this. We have recently acquired an Agilent Offgel fractionator. This instrument can separate either intact proteins or protease fragments into as many as 24 fractions based on isoelectric point. After a quick clean-up step, the fractions are ready for analysis. A second method for in-solution isoelectric focusing of proteins is use of our Bio-Rad microRotofor Cell. This method produces 10 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 by other methods.

2-D Gel Electrophoresis

Two-dimensional gel electrophoresis is an excellent way to separate and visualize as many as 2000 proteins in a sample. Replicate gels for control and experimental samples can be run simultaneously and the intensity of each spot on the gels can be quantified by our excellent gel analysis software. Once proteins of interest are found, they can be excised and identified by MS.

For 2-D electrophoresis, separation in the first dimension is performed by isoelectric focusing on immobilized pH gradient (IPG) strips which vary in length from 7 to 24 cm. Our lab contains 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. The second dimension is run on SDS gels. Gels can be stained by colorimetric or fluorescent stains or transferred for use in Western blotting.

Equipment for Electrophoresis and Transfer

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.

Staining and Imaging

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.

Software Analysis and Spot Cutting

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.

What is DIGE?

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.

Enzymatic Protein Digestion

Before being analyzed by mass spec, most proteins must be broken into smaller fragments. This is accomplished by digestion with a protease, usually trypsin, but may be another proteases of your choice.

Requirements: >100 fmol protein, may be a protein in solution, a gel slice or spot that is visible by coomassie staining.

In the Nevada Proteomics Center, trypsin digestion is performed either by a manual protocol, which is slow but quite effective, or by use of the very rapid Perfinity Flash Digest system.

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