The distance of purified protein=3.9 cm
Molecular weight of purified protein= -0.1872*3.9 + 2.1632
= 1.43312
Antilog of 1.43312= 101.43312
= 27.11kDa
Results and Discussion
Polyacrylamide gel: This gel is used as a supporting medium. It is the composition of two substances: acrylamide and bisacrylamide. When these two are combined a porous network is formed. The former determines the average polyacrylamide chain length. The latter determines the extent of the cross linking. The concentration of gel and the pore size is inversely proportional. To prepare this gel; we do not need to boil and then cool as we did to agarose. We need some assistant chemicals to prepare it. The chemicals are TEMED and APS. The preparation order is first the APS and then TEMED, to start polymerization the mixture has to be stirred.
Tetramethyletyl Diamine (TEMED): This chemical is the initiator of the polymerization of polyacrylamide.
Ammoniumpersulfate (APS): This is the catalyst of the reaction.
Sodium dodecyl sulphate (SDS) and 2-Mercaptoethanol: SDS is an anionic detergent and mercaptoethanol is a reducing agent. SDS binds to the protein, causing it to unfold, whereas the reducing agent reduces the intramolecular and intermolecular disulfide bonds. Therefore the molecule will be surrounded by a negative charge and will migrate to positive electrode due to its molecular weight.
Stacking and Separating Gels: These two are actually polyacrylamide with different pore sizes. The former is highly porous, denaturation of the protein takes place here. It consists of 4% acrylamide, 2.7% bisacrylamide, stacking buffer (tris-HCl), SDS, water, ammonium per sulphate and TEMED. The latter has smaller pores; it sieves the negatively charged proteins on the basis of weight. It contains all of what stacking gel contains, it only has running buffer (Tris-HCl) instead of stacking buffer.
Loading Buffer (or Tracking Dye): The dye used here is bromophenol blue. It increases the density of the sample and ensures that the sample goes in the gel. Also it migrates to the anode faster than proteins and shows the termination of electrophoresis. At last it gives colour to the sample, making loading of the sample into wells easier.
Staining Dye: Dye used on this experiment is coomassie blue R250. It binds not only to proteins but also all to the gel. The gel must stay in the dye for 4-8 hours in order to be stained successfully. After this process the gel have to be put into destaining solutions in order to get rid of the dye on the gel(but not on the proteins).
SDS is chosen in this system because it binds to most proteins in a constant fashion (about 1.4 grams of SDS per gram of protein) and also masks any charge of protein by forming large anionic complexes. SDS also disrupts any hydrogen bonds, blocks many hydrophobic interactions and partially unfolds the protein molecules minimising differences based on secondary or tertiary structure. The reducing agent mercaptoethanol is also included in order to totally unfold proteins and detach subunits to ensure a separation based exclusively on molecular weight.
The molecular weight of the molecule is inversely proportional to its mobility. Molecular weight of the protein can be estimated directly from a plot of the log of molecular weight versus mobility.
In our experiment we achieved a good result. The proteins were clearly seen and also we could calculate the molecular weight of our purified protein. SDS PAGE is an excellent system for producing individually purified proteins. But on the other hand it has some limitations. Proteins analysed are not easily recoverable. It is possible to remove them from the gel using a technique called electrophoresis or to digest the proteins within the gel using a protease enzyme. Also, proteins often react with the gel matrix. This leads to different amounts of acrylamide-protein polymers which makes identification of protein masses difficult.