Protein Purification II -Kevin Ahern's BB 450 Lecture #8 2016
Contact me at kgahern@davincipress.com Facebook friend me at https://www.facebook.com/kevin.g.ahern Protein Purification/Characterization II 1. PolyAcrylamide Gel Electrophoresis (PAGE) and agarose gel electrophoresis are means of separating molecules on the basis of size. In these techniques, a 'gel' is made that consists of strands of polyacrylamide or agarose that form a sort of 'mesh'. The more polyacrylamide or agarose there is and the more the strands are cross-linked, the harder it is for molecules to pass through it. Electric fields are used to separate macromolecules by size. The sample is loaded on the top of the gel and electrical current is passed through it such that the bottom electrode is positive and the top one is negative. Negatively charged molecules at the top of the gel are driven away from the top towards the bottom. Small molecules make their way through the gel fastest and big molecules travel more slowly. The key to gel electrophoresis is to have all of the molecules being separated have a negative charge. 2. Nucleic acids (which are negatively charged and 'rod-like' in shape) can be separated by PAGE or agarose gel electrophoresis readily without additional modification. Proteins, however are usually globular in their native state and, without other modification, may be negatively, positively OR neutrally charged. A modification of PAGE called SDS-PAGE is used to separate proteins. In this method, the detergent sodium dodecyl sulfate (SDS) is add to the protein mixture, causing the proteins to denature, assume rod-like shapes, and be coated with the negative charge of the SDS. Consequently all proteins in the mixture obtain a negative charge and can be separated just like DNA. 3. Isoelectric focusing is a technique that separates molecules on the basis of their pI (pH at which their net charge is zero). It is performed in tubes containing special compounds (polyelectrolytes) that migrate to specific points in the tube when in the presence of an electric field. This effectively creates a pH gradient from one end of the tube to the other. If proteins are added to the tube as the gradient is getting established, they will migrate to the point in the tube where the pH corresponds to their pI and they will migrate no further, since they will have a charge of zero. 4. 2D gel electrophoresis is a powerful tool for proteomics that combines the techniques of isoelectric focusing with SDS-PAGE. In this method, proteins are first separated according to their pI by isoelectric focusing. Then the tube from the isoelectric focusing is mixed with SDS and applied to the top of an SDS-PAGE gel and the proteins are separated by size. The result is a two dimensional separation of virtually every protein in the cell. 5. The intensity of each spot in the gel is a function of the quantity of protein - darker spots correspond to more protein. These protein 'spots' can be cut out of the gel, the protein can be extracted and then it can be characterized by other means to identify it, so the identity of each spot is known. 6. If one colors proteins from two different cells differently (orange from one, blue from another, for example), mixes them and performs the same analysis, orange spots will correspond to proteins in the first cell, but not the second. Blue spots correspond to being present in the second cell, but not the first. Black spots correspond to an equal mixture of the protein in each cell. 7. Microarrays are tools of transcriptomics - simultaneous analysis of all of the mRNAs of a cell or tissue. 8. In microarrays, a DNA fragment corresponding to the mRNA of very gene in a genome is made. 9. A grid on a slide is created with each spot on the grid being the place where thousands of copies of the DNA for each gene is covalently linked. Thus, spot one has thousands of identical DNA copies of a specific gene and spot 2 has thousands of identical DNA copies of a separate gene. 8. Each gene in the genome is represented by one spot. 9. mRNAs from two different cell types are isolated and DNA copies (called cDNAs) are made of each (DNA is easier to work with than RNA, hence the coversion to DNA). 10. The cDNAs are labeled with a color corresponding to the cell it came from - green from one, red from the other. 11. The cDNAs are mixed and then poured onto the slide with the microarray. Hybridization is allowed to occur and then unhybridized strands are washed away. 12. Upon analysis in a microscope, the color and intensity of each spot is determined. Red corresponds to cDNA (mRNA) from one cell, but not the other and green corresponds to cDNA from the other cell, but not the first one. An equal mix of cDNA from each cell yields a yellow color and the intensity of each color is an indication of the amount of mRNA/cDNA present in each cell.
Contact me at kgahern@davincipress.com Facebook friend me at https://www.facebook.com/kevin.g.ahern Protein Purification/Characterization II 1. PolyAcrylamide Gel Electrophoresis (PAGE) and agarose gel electrophoresis are means of separating molecules on the basis of size. In these techniques, a 'gel' is made that consists of strands of polyacrylamide or agarose that form a sort of 'mesh'. The more polyacrylamide or agarose there is and the more the strands are cross-linked, the harder it is for molecules to pass through it. Electric fields are used to separate macromolecules by size. The sample is loaded on the top of the gel and electrical current is passed through it such that the bottom electrode is positive and the top one is negative. Negatively charged molecules at the top of the gel are driven away from the top towards the bottom. Small molecules make their way through the gel fastest and big molecules travel more slowly. The key to gel electrophoresis is to have all of the molecules being separated have a negative charge. 2. Nucleic acids (which are negatively charged and 'rod-like' in shape) can be separated by PAGE or agarose gel electrophoresis readily without additional modification. Proteins, however are usually globular in their native state and, without other modification, may be negatively, positively OR neutrally charged. A modification of PAGE called SDS-PAGE is used to separate proteins. In this method, the detergent sodium dodecyl sulfate (SDS) is add to the protein mixture, causing the proteins to denature, assume rod-like shapes, and be coated with the negative charge of the SDS. Consequently all proteins in the mixture obtain a negative charge and can be separated just like DNA. 3. Isoelectric focusing is a technique that separates molecules on the basis of their pI (pH at which their net charge is zero). It is performed in tubes containing special compounds (polyelectrolytes) that migrate to specific points in the tube when in the presence of an electric field. This effectively creates a pH gradient from one end of the tube to the other. If proteins are added to the tube as the gradient is getting established, they will migrate to the point in the tube where the pH corresponds to their pI and they will migrate no further, since they will have a charge of zero. 4. 2D gel electrophoresis is a powerful tool for proteomics that combines the techniques of isoelectric focusing with SDS-PAGE. In this method, proteins are first separated according to their pI by isoelectric focusing. Then the tube from the isoelectric focusing is mixed with SDS and applied to the top of an SDS-PAGE gel and the proteins are separated by size. The result is a two dimensional separation of virtually every protein in the cell. 5. The intensity of each spot in the gel is a function of the quantity of protein - darker spots correspond to more protein. These protein 'spots' can be cut out of the gel, the protein can be extracted and then it can be characterized by other means to identify it, so the identity of each spot is known. 6. If one colors proteins from two different cells differently (orange from one, blue from another, for example), mixes them and performs the same analysis, orange spots will correspond to proteins in the first cell, but not the second. Blue spots correspond to being present in the second cell, but not the first. Black spots correspond to an equal mixture of the protein in each cell. 7. Microarrays are tools of transcriptomics - simultaneous analysis of all of the mRNAs of a cell or tissue. 8. In microarrays, a DNA fragment corresponding to the mRNA of very gene in a genome is made. 9. A grid on a slide is created with each spot on the grid being the place where thousands of copies of the DNA for each gene is covalently linked. Thus, spot one has thousands of identical DNA copies of a specific gene and spot 2 has thousands of identical DNA copies of a separate gene. 8. Each gene in the genome is represented by one spot. 9. mRNAs from two different cell types are isolated and DNA copies (called cDNAs) are made of each (DNA is easier to work with than RNA, hence the coversion to DNA). 10. The cDNAs are labeled with a color corresponding to the cell it came from - green from one, red from the other. 11. The cDNAs are mixed and then poured onto the slide with the microarray. Hybridization is allowed to occur and then unhybridized strands are washed away. 12. Upon analysis in a microscope, the color and intensity of each spot is determined. Red corresponds to cDNA (mRNA) from one cell, but not the other and green corresponds to cDNA from the other cell, but not the first one. An equal mix of cDNA from each cell yields a yellow color and the intensity of each color is an indication of the amount of mRNA/cDNA present in each cell.