1.1 Lab Techniques Flashcards

Question

Describe SDS-PAGE Electrophoresis

Answer 1

SDS-PAGE separates proteins by size alone. The proteins are denatured by heat in the presence of a detergent which causes them to unfold. This results in a linear protein with an equally negative charge. When these gels are run only size of the molecule becomes a consideration.

Answer 2

Isoelectric point (IEP) is the pH at which a soluble protein has no net charge and will precipitate out of solution If the solution is buffered to a specific pH only the protein(s) which have an IEP of that pH will precipitate

Answer 3

Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its isoelectric point in the pH gradient because it has no net charge.

Answer 4

Antibodies are Y-shaped globular proteins produced by B lymphocytes as part of an immune response.

Answer 5

Immunoassay techniques

Answer 6

Used in the detection and identification of specific proteins

Answer 7

Each B lymphocyte produces one specific antibody that binds to one specific antigen. This binding helps to render an antigen harmless. Immunoassay techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies

Answer 8

Immunoassay techniques (such as ELISA) involve the use of monoclonal antibodies that have an enzyme attached. This reporter enzyme catalyses a colour change reaction that is used to detect and quantify the presence of a specific antigen. If the antigen is present, the antibody binds allowing the reporter enzyme to produce a coloured product; providing visual confirmation of the antigen’s presence. If the antigen is not present then the antibody can not bind and the enzyme is washed away before a reaction can take place. Blood samples can be screened for either antigens or antibodies themselves using this technique. Chemical labels other than reporter enzymes include chemiluminescence and fluorescence.

Answer 9

Western blotting is a technique for identifying specific proteins that have been separated using SDS-PAGE electrophoresis

Answer 10

The proteins are blotted from a gel onto a solid medium. It is then flooded with fluorescent labelled monoclonal antibodies (several different ones can be used simultaneously if they have different coloured markers). Once the antibodies have bound to their target proteins, the excess is washed away. When exposed to particular wavelengths of light the antibodies fluoresce allowing the precise location of target proteins to be identified.

Answer 11

Bright-field microscopy is commonly used to observe whole organisms, parts of organisms or thin sections of dissected tissue or individual cells. Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

Answer 12

Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells. It involves the sterilisation of equipment and culture media by heat or chemical means and therefor the subsequent exclusion of contaminants.

Answer 13

A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients. Many different types of culture media exist that promote the growth of specific types of cells and microbes. The type of media required should be investigated prior to culturing. Animal cells are grown in medium containing growth factors from serum.

Answer 14

proteins that promote cell growth and proliferation

Answer 15

A cell line is a genetically uniform cell culture developed from a single cell

Answer 16

In culture the cells soon use up the nutrients in the medium so to keep the cloned cell line alive some must be sub cultured into a fresh culture flask. Newly created animal cell lines tend to die after a finite number of divisions (about 60 – the Hayflick limit). This reduces the length of time a primary cell line cultured can be maintained. However, this limit does not exist in cancer cell lines, which are immortal and can be sub cultured indefinitely

Answer 17

Plating out of a liquid microbial culture on solid media allows the number of colony forming units to be counted and the density of cells in the culture estimated. Even then serial dilution is often needed to achieve a suitable colony count

Answer 18

To estimate cell numbers in a liquid culture

Answer 19

A haemocytometer is a graduated microscope slide used to count cell density.

Answer 20

The coverslip of a haemocytometer slide is designed to sit a known distance from the slide. This means that the viewer is looking through a known volume of medium. By counting the number of cells in a particular area of the grid, a cell density can be calculated

Answer 21

Using a haemocytometer to make total cell counts or if a vital stain is used then the living cells can be distinguished.

Answer 22

An example of a vital stain is methylene blue; when yeast is viewed under the microscope at high power using this stain the vital or living cells are colourless, whereas dead cells are blue.