Experiment 3: SDS PAGE I : Gel Preparation

-PurposeTo prepare SDS PAGEs gels (stacking gel and seperating gel) for further experiment. =Introduction= SDS-PAGE is a molecular biology technique used to separate proteins accordingly by size. SDSPAGE also can separate DNA and RNA molecules. In what is probably the most powerful technique for resolving protein mixtures, proteins are exposed to ionic detergent SDS (sodium dodecylsulfate) before and during gel electrophoresis. SDS denatures proteins, causing multimric proteins to dissociate into their subunits, and all polypeptide chains are forced into extended conformations with similar charge:mass ratios. SDS treatment therefore eliminates the effects of differences in shape so that chain length, which reflects mass, is the sole determinant of the migration rate of proteins in SDS- polyacrylamide electrophoresis. Even chains that differ in molecular weight by less than 10 percent can be separated by this technique. Moreover, the molecular weight of a protein can be determined by comparison to a protein ladder or molecular weight ladder which is run on the same gel. The basic idea of separating gel is to cause particles of varying sizes to move through a gel made up of some physical 'mesh'. Common biological gels are made of Agarose proteins (for separating nucleic acids) or Acrylamide/bisAcrylamide 'mesh' for separating proteins. Smaller molecules can move through the network faster, and so will move farther in a particular time period (minute, half-hour, hour, whatever). So, if you run a gel for 30 minutes or so, you will have a distribution of sizes, with the smallest pieces out in front, and the larger ones progressively further behind. Often you will have these molecules moving in 'bands', which are clumps of lots of a molecule which are the same size. Usually, you get the proteins or nucleic acids to move by running a current across the gel, as these molecules have a slight electrical charge. If you also run a set of comparable molecules of known sizes (usually called a 'size standard') then you have something to compare your results to so you can tell what size a particular band is. The stacking gel is a large pore PAG (4%T). This gel is prepared with Tris/HCl buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer (Tris/Glycine). These conditions provide an environment for Kohlrausch reactions determining molar conductivity, as a result, SDS-coated proteins are concentrated to several fold and a thin starting zone of the order of 19 m is achieved in a few minutes. This gel is cast over the resolving gel. The height of the stacking gel region is always maintained more than double the height and the volume of the sample to be applied.this is based on [isotachophoresis] that is glycine from electrophoresis buffer have lower electrophoretic mobility than protein-sds complex which is less mobile than cl- ions.giycine-<sds protein<cl-.if anyone of them moves faster there will be a break in the electric circuit.to move in uniform speed glycine should be in a region of higher (electic) field strength.field strength is inversly propotional to conductivity which is directly propotional to concentration.so adjust acoordingly and protein is stacked between glycine and Cl but at seperating gel there is higher pH 8.8 they travel according to their speed, so seperating proteins.

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and nondisulfidelinked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide. SDS bind in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run. Acrylamide is a chemical that is used to make polyacrylamide materials. Polyacrylamide is used in the treatment of drinking-water and waste water where it is used to remove particles and other impurities. It is also used to make glues, paper and cosmetics. Polyacrylamide materials contain very small amounts of acrylamide. Acrylamide is also produced in some foods prepared at high temperatures, for example in frying and roasting, so common food sources containing acrylamide include french fries, chips, biscuits, bread and coffee beans. Acrylamide is known to cause cancer in animals. Also, certain doses of acrylamide are toxic to the nervous system of both animals and humans.The levels of acrylamide in food stuff is very very smal so risks would be associated to long term exposure, therefore like all recommendations a balanced and varied diet reduces risks considerably by not repeatly eating the same thing over and over. (N, N, N, N-tetramethylethylenediamine) Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium persulfate and the quaternary amine, N,N,N,N-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. Increasing the amount of APS and TEMED results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount of initiators shows the reverse effect. The lowest catalysts concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used, approximately in equimolar concentrations in the range of 1 to 10 mM.

=Equipments=

SDS PAGE prep kit Test tube Deionized Water Distiled water SDS (10%)

APS (10%) Acrylamide TEMED Isopropanol Bisacrylamide

Tris (1.5M) Micropipette and tips Pipette Ethanol (70%)

-Procedure-

Clean the glass plates with distilled water, then use ethanol. Wait them to dry. Add 1.7 ml deizonized water to the test tube. Add 2ml Acr/Bis onto it. Add 1.25 ml of 1.5M Tris. To mix well, vibrate the tube gently. Add 50 l SDS (10%). Add 50 l APS added. Add 2.2 l of TEMED. Shake the tube gently. Wait the mixture to become rigid.

Note : The process above is belong to seperating gel. Yet this process should be apply for stacking gel too, with different rate.* *This data is in appendix part.

=Appendix=

Stacking Gel (3ml) dI H2O Acr/Bis Tris(1.5M)(pH6.8) SDS (10%) APS (10%) TEMED Separating Gel (5ml) dI H2O Acr/Bis Tris(1.5M)(pH8.8) SDS (10%) APS (10%) TEMED

2.2 ml 1 ml 0.75 ml 30 l 30 l 3l

1.7 ml 2 ml 1.25 ml 50 l 50 l 2.2 l

fig 1 : Components of SDS-PAGE

=Discussion= This experiment is the first part of SDS PAGE experiment. This part targeted to prepare stacking and seperating gels. Seperating gel and stacking gel are made with using the same materials but with different rates. Seperating gel is prepared first because staking gel take upper place. Ingredients of seperating gel are deionized water, acrylamide,bisacrylamide, tris, SDS ,APS and temed. Acrylamide is a chemical that is used to make polyacrylamide materials. Bisacrylamide is
used cross linking agent for polyacrylamide gels. Tris performs as buffer regulate pH level-.SDS is used to denature protein. SDS is a detergent. APS is a source of free radicals and is often used as an initiator for gel formation. Temed is not indispensable, because temed makes this process easier and faster.
These steps were what we followed in lab and in the end we got a successfull yield seperating gel-. We did not made a stacking gel but as known two of them are not different that much. Seperating gel has much pores when stacking gel has less pores. This is why stacking gel is used to get protein in a line, but seperating gel is used to seperate proteins. In conclusion this process is an essential part of SDS-PAGE, and also SDS-PAGE is a significant method in biochemistry that is used to determine what size of proteins there are in a sample. To know this method will help us for several study.