Question

Describe these two techniques: co-immunoprecipitation and sandwich ELISA. For each technique, be sure to explain its purpose and describe the main steps involved in carrying out the technique.

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Answer 1

Co-immunoprecipitation

Co-immunoprecipitation (co-IP) is a technique to identify physiologically relevant protein–protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.

The general steps are as follows:
1. Lyse your Cells
2. Add your Antibody
3. Add the Protein A/G Beads
4. Incubate
5. Collect
6. Wash the Beads
7. Elute your Protein(s)
8. Detect your Protein(s)
9. Study the Interaction With Surface Plasmon Resonance Applications.

Purpose:
Immunoprecipitation method was originally invented as an alternate to affinity chromatography for small scale protein production. This method is a popular choice for identifying and separating protein with low abundance. Co-immunoprecipitation is a well known and popular method for studying protein-protein ineraction invitro. The main applications are

• Prove interaction between two proteins
• Identifies a new protein by using a known one.

Sandwich ELISA
Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection. Moreover, many commercial ELISA pair sets are built on this sanwich ELISA. The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody).

The steps of sandwich ELISA are as follows:

1. Prepare a surface to which a known quantity of capture antibody is bound.
2. Block any nonspecific binding sites on the surface.
3. Add antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. A specific antibody is added, and binds to antigen (hence the ‘sandwich’: the Ag is stuck between two antibodies);
6. Add enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody’s Fc region (non-specific).
7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
8. Add substrate that is converted by the enzyme into a color or fluorescent or electrochemical signal.
9. Measure the absorbeance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

Purpose:
The Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most efficient laboratory procedures used in detecting the presence and measuring the concentration of a target antigen in a completely unknown sample.
They are considered highly sensitive, specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests.