Weaver eoc answers 05

Weaver eoc answers 05 - Answers to Weaver end of chapter...

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Answers to Weaver end of chapter questions Chapter 5 Molecular Tools for Studying Genes and Gene Activity 1. 2. SDS, sodium dodecyl sulfate, is a detergent used in SDS polyacrylamide gel electrophoresis. SDS denatures the proteins and separates the subunits of complex proteins. In addition, it coats all the polypeptides with a negative charge so that they migrate according to their molecular masses and not their natural charge. 3. SDS-PAGE and modern two-dimensional gel electrophoresis both separate proteins according to their molecular masses. However, in two-dimensional gel electrophoresis, the proteins are subjected to isoelectric focusing prior to separation by SDS-PAGE. In the isoelectric focusing process, the proteins are electrophoresed in a pH gradient. Proteins will stop migrating when they reach their isoelectric point. The gel from the tube is then placed on the top of an SDS- PAGE gel and subjected to separation according to molecular mass. Two dimensional gel electrophoresis therefore separates proteins based on both their mass and isoelectric point. SDS-PAGE separates proteins solely based on their molecular mass. 4. Ion exchange chromatography separates proteins based on their net charge. In this procedure a complex protein mixture is loaded onto a charged resin. In the case of anion exchange chromatography this is a positively charged resin. Solutions of increasing ionic strength are passed over the column and the ions in these solutions compete with the proteins for binding sites on the resin. Proteins with less charge are eluted at lower ionic strength than those with a greater charge. 5. Gel filtration chromatography separates protein according to their physical dimensions. It involves passing the proteins through a porous resin in a column. The resin has holes of a + - DNA loaded in wells DNA runs towards The positive pole 1200 bp 600 bp 150 bp 1
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defined size. Smaller proteins emerge more slowly through the column than larger ones because their smaller dimension allows them to pass into the holes in the resin giving them a longer path to travel. Larger proteins, on the other hand, will not be able to enter the beads and will emerge more rapidly. Intermediate sized proteins will enter some beads and not others and will emerge after an intermediate period of time. 6. Autoradiography and phosphorimaging are both techniques that allow visualization of a radioactive signal immobilized on a membrane. Both detect the β-particles emitted from the isotopes commonly used in molecular biology. In the case of autoradiography, the emitted particles expose the emulsion on an x-ray film, leaving dark bands. Autoradiography is limited in its ability to quantify the amount of isotope exposing the film. The reason for this is that the response of film to radiation is non-linear and saturation of the signal is often a problem. For example, a sample with say 10,000 dpm (disintegrations per minute) may expose the emulsion on the film to its maximum capacity and therefore another sample with 50,000 dpm will give a
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Weaver eoc answers 05 - Answers to Weaver end of chapter...

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