Gel Electrophoresis Gel electrophoresis is a technique that has been around for some time. It is simply a method used to separate differently sized molecules of DNA, RNA, or protein. Gel electrophoresis has wide applications. The principle behind electrophoresis is rather simple. A gel is made of starch, agarose (for DNA or RNA), or polyacrylamide (for proteins). The gel is emersed in a buffer solution in order to maintain pH levels and to allow molecules (DNA, RNA, or protein) to move through the gel. The samples of protein, DNA or RNA are carefully loaded into wells (or "slots") of the gel. An electric current is applied which forces the protein, DNA, or RNA to move through the gel -- larger molecules move slower than smaller molecules, therefore molecules separate based on size (or charge). At some point, the electric current is stopped (to prevent the samples from running off the gel). Usually, a lane with a standard marker (which is commercially available) is included, so that the relative sizes of samples can be identified. For example, the sizes of DNA and RNA markers are in bases (b) or kilobases (kb), while proteins markers are in kiloDaltons (kDa; one Dalton is approximately equal to the mass of one hydrogen atom). Sometimes commercially available proteins are also run in a gel alongside samples, which can be used to identify the sizes of particular proteins in a sample. For example, in the work that I do, I usually include lanes with spectrin (220 kDa and 240 kDa, isolated from human red blood cells) and alpha-actinin (100 kDa, isolated from chicken gizzard) to determine whether a particular band (after immundetection) in a sample is comparable in size to these known proteins. After gel electrophoresis, a transfer apparatus (or "blotting" apparatus) is set up so that the samples in the gel will transfer and become embedded in a "filter" or membrane (I use PVDF membranes). This transferring process to the membrane maintains the relative positions of the samples that were separated in the gel. Diagrams of the electrophoretic and transfer systems can be found in any first year biology textbook, so I won't provide any here except for the simple schematic shown below (Fig-1). In the following schematic, the negative and positive ends on the gel are indicated, and the flow of electricity moves from negative to positive (Fig-1, arrow in lane 1) which forces proteins to move through the gel. Different sizes of proteins move at different speeds (i.e. larger proteins move slower than smaller sized proteins). A gel can have several lanes (the one in the diagram shows four lanes, labelled at the top of the gel) -- the system I use has ten lanes -- so that multiple samples can be run simultaneously. Protocols may differ slightly with the different equipment that are commercially available (the system I use is from Bio-Rad). Regardless of this matter, I have included a protocol that I use for gel electrophoresis and transfer, below. Gel Electrophoresis (SDS-PAGE) Stock Reagent dH2O 1.5 M Tris-HCl pH 8.6 10% SDS 30% Acrylamide 10% APS TEMED Separating Gel (7.5%) 9.7 ml 5.0 ml 200 ul 5.0 ml 100 ul 10 ul Stock Reagent dH2O 0.5M Tris-HCl pH 6.8 10% SDS 30% Acrylamide 10% APS TEMED Stacking Gel 6.1 ml 2.5 ml 100 ul 1.33 ml 50 ul 10 ul Note that the pH values for the Tris-HCl are different for separating and stacking gels. Also note that the final volumes above are enough for making two gels. The percentage of the gel (i.e. 7.5%) can be modified by varying the volumes of acrylamide and water added. Also, the voltage used (shown below) is for two gels (i.e. if only one gel was run at a time, then the voltage would have to be reduced to prevent overheating -- otherwise the gel would melt). Loading Samples and Running the Gel Ratio = 2 (loading buffer) : 1 (sample) Heat for 5 minutes, then cold shock (~5 min on ice) and spin down 1 x Running Buffer (80 ml of 5x RB plus 320 ml dH2O) Run at 80 volts (50 amps) for stacking gel (~20 min) Then increase to 160-170 volts (60-80 amps) Loading Buffer (LB) (10ml final volume): 0.05 M Tris-HCl, pH 6.8 [1 ml of 0.5 M Tris] 0.1 M DTT [2 ml 0.5 M DTT] 2% SDS [2 ml of 10% SDS] 0.1 % Bromophenol Blue [0.01 g] 10% Glycerol [1 ml] 5 x Running Buffer (RB) (600ml final volume): 9 g Tris-base 43.2 g Glycine 3 g SDS dilute 80 ml of 5 x buffer with 320 ml of dH2O to make 1 x buffer Gel Transfer to PVDF Membrane Set up transfer (as shown in Bio-Rad manual) using PVDF membrane add transfer buffer (also use a magnetic bar) Run it at 30 volts (no more than 33) O/N (overnight) in cold room, with stirring When complete, dry membranes on blotting paper Transfer Buffer: 3.03 g (25mM) Tris-base 14.4 g (192 M) Glycine 200 ml (20%) methanol add dH2O to 1 L, check for pH 8.1 to 8.4 prechill before use SITE MAP EVOLUTION INDEX. This page hosted by Get your own Free Home Page