1.1 Detecting proteins using antibodies

Question 1

Immunoassay techniques

Answer 1

Used to detect and identify specific proteins. These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies.
An antibody specific to the protein antigen is linked to a chemical ‘label’. The label is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.

Question 2

Western blotting

Answer 2

Technique used after SDS-PAGE electrophoresis. The separated proteins from the gel are transferred (blotted) onto a solid medium.

Question 3

Specific antibodies

Answer 3

The proteins can be identified using specific antibodies that have reporter enzymes attached. In some cases the assay uses a specific antigen to detect the presence of antibodies.

Question 4

Bright-field microscopy

Answer 4

Commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.

Question 5

Fluorescence microscopy

Answer 5

Uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

Question 6

ELISA techniques

Answer 6

(enzyme-linked immunosorbent assay) - analytical technique which uses antibodies to detect presence of an antigen within a solution. 3 forms direct, indirect, sandwich.

A colour producing substrate is added to the well and a colour is allowed to develop. Colourless means negative result (protein not present), or colour change show a positive result (protein is present).

Question 7

ELISA: direct

Answer 7

The antigen is allowed to bind to the surface of a multiwell plate. A primary antibody, linked to a reporter enzyme, is added to the well and binds to the antigen.

Question 8

ELISA: indirect

Answer 8

The antigen is allowed to bind to the surface of a multiwell plate. A primary antibody is added to the well and allowed to bind to the antigen. A secondary antibody linked to a receptor enzyme, is then added, which binds to the primary antibody.

Question 9

ELISA: sandwich

Answer 9

A capture antibody is bound to the surface of a multiwell plate. The antigen is added and allowed to bind to the capture antibody. A primary antibody, which binds to the antigen, is added to the well. A secondary antibody, linked to a receptor enzyme is then added, which binds to the primary antibody.