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●Gene fusion technique, GST, MBP, etc. ○Gene fusion technique: the gene encoding the protein of interest is fused to a second gene encoding a protein that is easily purified by affinity chromatography. The second protein is the “tag”.○This encodes a large protein that consists of the protein of interest fused to the tag protein. And these 2 proteins are often separated by a linker of amino acids, this provides a protease cleavage site that can be used to separate the tag from the protein of interest.○Examples of tags:■GST (glutathione-S-transferase): Used to purify GST by binding it to bound GSH and eluted with GSH.■MBP (maltose-binding protein):Binds to column with bound maltose and eluted with maltose■Enhance solubility: GST,Trx,MBP,NusChapter 9 - SDS-PAGE, Protein Visualization, and Band Quantification●SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis):○This was developed to allow proteins to separate on molecular weight alone. By adding Sodium Dodecyl Sulfate, a detergent, amino acids are covered in an overwhelming negative charge, the charge to mass ratio is identical for all proteins. ○Purpose:To determine the approximate molecular weight of the protein of interest ○SDS and heat:Breaks weak interactions and causes the protein to unfold and linearize ○β-Me:Breaks the disulfide bonds. ●Mechanism of separation, direction of current/movement of bands, large vs. small proteins ○Since all proteins are now covered in a negative charge, they will migrate to the positive electrode (anode) based on size alone, the smaller proteins will migrate the fastest, while the larger proteins will migrate the slowest in the gel. ●Gradient gels: purpose, differences ○Stacking gel:is cast on top of the resolving gel, has about 5% acrylamide. Samples migrate quickly creating a narrow band before reaching the resolving gel so proteins are presented at the same time ○Resolving Gel:has about 10-15% acrylamide (higher than stacking) and is where the actual separation of proteins based on the molecular weight takes place ●SDS-PAGE vs. SEC (how do large proteins migrate in each?)
○On SDS-PAGE, smaller molecules migrate the fastest vs on SEC smaller molecules migrate the slowest; large proteins are higher on SDS PAGE gel while they elute first in SEC. ●Purpose of stacking gel, why it is necessary in SDS-PAGE (and not DNA gels) ○In SDS-PAGE, the proteins are inserted in line with the direction of migration, whereas on an agarose gel, samples are loaded perpendicular to this migration. Therefore a stacking gel is unnecessary since the samples migrate in a tight band w/o the need to concentrate it. ●Visualization methods, quantification of protein bands, molecular weight determination ○Coomassie brilliant blue:binds to proteins, most common, this technique requires fixing proteins to the gel to ensure they don’t migrate out of the gel.