MCB 250 1st Edition Lecture 9Outline of Last Lecture1. Protein Folding2. Chaperones3. GroE4. Peptide Bonds5. Posttranslationally Modified6. Denaturing ProteinOutline of Current Lecture 7. Protein purification8. Column ChromatographyCurrent Lecture1. Protein purification:- TO study proteins, you need large amounts (mg)- Various techniques are used to separate proteins based on their properties, usually chromatography:o Based on size, charge, hydrophobicity, specific binding2. Column Chromatography- Start with a crude extract of proteins and the matrix will separate the protein - Can separate based on charge, shape, and specific binding- Separation based on charge: ION EXCHANGEo The protein is charged and will stick to the beads with the opposite charge or repel with the similar charge. In example, use negatively charged beads. The mixture of protein will be dumped in and positive charged proteins will stick to the negative beads. The negative proteins wont stick and will escape to the bottom. Dump more elution in the solution to get positive proteins to come out after the negative has come out. Does not split based on shape or size.- Separation based on size: SIZE EXCLUSION OR GEL FILTRATIONo The beads contain pores. The small proteins go through the beads and explore longer through it than the larger proteins. Very large molecules can’t enter the beads. Medium take some space to explore. Small proteins slow as they go through the column- Separation based on Affinity: AFFINITY CHROMATOGRAPHYThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o HISTAGS: DNA encoding protein you want to purify and place it near a plasmid vector with sequence of 6 histidines and the gene of interest is inframed by ito The plasmid will produce more proteins and you want to purify it with its C-terminalo Multiple histidines in a row can coordinate with nickelo Take all the proteins and the protein of interest has the his tag in it and will bind to nickel. Put all the crude extract into the Ni column and the his tags will bind to the Ni. Then pour eluent that competes with the his-tags for the Ni, which resultsin the release of tagged protein. The protein of interest will fall
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