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Lecture 2.3

Element 2- Biochemistry

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.
Created by: macdonkr
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