Lecture 10Outline of Last Lecture I. Protein PurificationA. Protein Purification MethodsII. Gel Filtration ChromatographyIII. Ion Exchange ChromatographyA. Monitoring ProgressIV. Gel ElectrophoresisA. Visualizing the Proteins Outline of Current Lecture I. Protein AnalysisA. Two-dimensional Gel ElectrophoresisB. Definition of Isoelectric PointII. Protein CleavageA. Definition of ProteasesIII. Examples of ProteasesIV. Protein SequencesCurrent LectureAs mentioned in the previous lecture, protein separation can be visualized and analyzed through gel electrophoresis. This can be done by using an electrical current to show the different protein bands. Another way is to do a 2 dimensional gel electrophoresis to separate the proteins. The first dimension uses isoelectric points to separate the various proteins. The isoelectric point is the pH at which the whole protein has a neutralnet charge. After this is done, the second dimension separates the proteins based on their sizes. The proteins can then be seen after staining the gel. This gel is normally hard to interpret and is very messy looking, with many bands. Proteins are hard to analyze when they are whole and intact. The proteins are often cut into pieces or fragments. The fragments can then by analyzed using mass spectrometry. The enzymes used to cut up proteinsare called proteases. Proteases are enzymes that cut the peptide bonds within proteins, to create fragments. Most proteases are non-specific and cannot be used for biochemistry. The useful proteases that biochemists use are specific. Trypsin is a protease that cleaves the peptide bond after lysine and arginine. Chymotrypsin is a proteasethat cleaves the peptide bond after tyrosine, phenylalanine, and tryptophan. Cyanogen bromide is the most common protease used. It will cleave the peptide bond after methionine. Sometimes other chemicals can be used as proteases. BCHM 309 1nd EditionUsing all three agents can help to piece a protein sequence back together. The overlapping set of peptide fragments formed gives the whole protein
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