click below
click below
Normal Size Small Size show me how
Lecture 2.3
Element 2- Biochemistry
Question | Answer |
---|---|
How does the gel used in electrophoresis work? | Long chains of poly acrylamide with bis-acrylamide forming cross- links create a mesh-like structure. The varying concentrations of the polymer form varying pore sizes and acts like a molecular sieve. |
How does gel-electrophoresis separate molecules? | When an electric field is applied, molecules move according to their charge density= overall charge/ molecular volume |
Do longer or shorter molecules move further in gel-electrophoresis? | Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. |
How does SDS-PAGE differ from gel-electrophoresis? | It makes all molecules have the same charge density by changing each molecules charge proportional to its size. Molecules are separated by MOLECULAR MASS rather than CHARGE DENSITY |
How does SDS-PAGE create a uniform charge density for all proteins in the sample? | SDS evenly coats proteins with negatively charged detergent molecules, meaning that the larger molecules have a larger negative charge as more SDS can attach. Hence charge density is the same. |
What does SDS-PAGE stand for? Which part is the detergent? | Sodium Dodecyl Sulphate- Polyacrymide Gel Electrophoresis. SDS is the detergent. |
Why is SDS added to the proteins before gel electrophoresis? | SDS is added to the proteins as a reducing agent to remove disulphide bonds |
What happens to the proteins after SDS is added in electrophoresis preparation? Why? | They are boiled= to denature them (quaternary structure disappears) and expose the internal hydrophobic sections onto which the aliphatic carbon dodecyl carbon chain of the SDS can hydrogen bond to. |
What is a aliphatic carbon? | An aliphatic compound is a hydrocarbon compound containing carbon and hydrogen joined together in straight chains, branched trains or non-aromatic rings |
What are isoenzymes? | Enzymes that are the same but are coded for by different genes. The primary structure is different but the 3D shape they fold into are very similar |
Are the functions of isoenzymes similar? | Yes their functions are the same due to the fact that their 3D structure is very similar but the responses are very slightly different (i.e. needs more /less substrate to function) |
What does ELISA stand for? | Enzyme-linkd immunoabsorbent assay |
What can ELISA be used to detect? | Immunological diseases eg HIV |
What is ELISA used for? | The process of detecting an antigen in a sample. |
What is Gel Electrophoresis used for? | Gel electrophoresis is a laboratory technique used to visualise DNA on a gel where the DNA molecules are separated according to their size. This can allow scientists to detect alterations in the DNA sequence which may cause a genetic condition. |
What is SDS-PAGE used for? | Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. |
How does ELISA work? | The antibody in the sample is associated with an enzyme and when the enzyme reacts with its subtract a colour change is observed. The amount of coloured product formed per unit time is proportional to the amount of antigen in the substance |
What are the steps of the process of ELISA? | 1. Antibody with associated enzyme added to test sample. 2. Antibody binds with antigen. 3. Unbound antibodies are removed, only the antibody/ enzyme complexes that are bound to the antigen left 4. Enzyme substrate added 5. Coloured product formed |
What happens if an extra antibody is involved in the process that binds to another antibody? | An antibody-antibody-enzyme comples is produced. Very useful when the amount of antigen you want to test for is very small. |