SDS-PAGE Separation of Proteins Produced by Beans During Development

Background Information
Sodium Dodecylsulfate (
SDS
, also called Sodium Lauryl Sulfate) is an anionic detergent which denatures proteins by wrapping around the peptide backbone. This results in two things 1) a net negative charge proportional to the length of the protein in question and 2) uniform rod shaped proteins. Because shape is not a factor, as all proteins exposed toSDSwill be rod shaped and charge is proportional to the length of each protein, it is possible to separate these proteins on the basis of size using gel electrophoresis.
In gel electrophoresis, molecules with a net electrical charge, in this case a negative charge due to the SDS, are placed in an electrical field. Under these conditions, the charged molecule moves toward the pole with a charge opposite to that carried on the molecule. In this particular case, the proteins move toward the positive pole as they carry a negative charge. Usually the molecules are forced to move through a gel matrix. As smaller molecules can move through the matrix faster than large molecules, the molecules become separated with fast moving bands of small molecules at the front and slow moving bands of larger molecules trailing behind.
Proteins produced during the process of seed germination in beans will be separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE in this laboratory). Beans will be allowed to germinate for different periods of time ranging from zero to ten days. They will then be ground up and the proteins extracted. The proteins will be separated on a polyacrylamide gel matrix.
Polyacrylimide is a polymer of acrylamide and N,N’-methylene-bis-acrylamide (bis). The bis provides the crosslinks between long polymers of acrylamide. As a consequence the pore size of the gel can be controlled by varying the ratio of acrylamide to bis. Typically, a ratio of 37.5:1 of acylamide to bis is used to separate proteins although it may be altered depending on the size of the proteins being separated.
Preparation of Germinated Beans
Beans will be started germinating 10 days before the lab and every day that follows, more beans will be started so that by the time lab is held beans will have been germinating for 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 days. Bean germination will be achieved by placing a wet paper towel in a glass culture dish then placing 6 - 8 beans on the towel. After placing another wet paper towel on top of the beans, a second culture dish should be placed on top of the first one to prevent excessive evaporation. Be sure to carefully mark each dish with the date that the seeds were started germinating.
Preparation of the Polyacrylamide Gel
Because acrylamide is a dangerous neurotoxin and is also a suspected carcinogen, great care should be exercised in its handling. Always wear gloves, and clean up spills immediately. Do not eat or drink anything in lab, or before washing your hands after lab. You will be using a premixed acrylamide solution that is not quite as dangerous as the dry powder, but should still be treated with caution
The type of gel that we will be casting is a discontinuous gel. In this type of gel, the part of the gel used for separation of the fragments is cast first, then a second gel called a stacking gel is cast on top. The staking gel causes proteins in dilute solutions to form a very sharp concentrated band before contacting the separating gel. This allows the loading of dilute protein solutions onto the gel while still producing sharp bands on the separating gel. ……………：