Lab 8 - Immunoprecipitation

Question 1

What are the tasks of this lab?

Answer 1

Immunoprecipitation, preparation of samples for SDS-PAGE, separation of proteins on SDS-PAGE, and electroblotting

Question 2

What applications are immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting routinely used for?

Answer 2

1) Determine the molecular weights of protein antigens
2) Study protein/protein interactions
3) Determine specific enzymatic activity
4) Monitor protein post-translational modifications
5) Determine the presence and quantity of proteins

Question 3

What does the IP technique enable?

Answer 3

The detection of low abundant proteins in mammalian cells which otherwise would be difficult to detect since this technique can concentrate proteins up to 10,000-fold-depending on the quality of antibody

Question 4

What is an antigen?

Answer 4

• Often a protein or peptide
• Foreign substance that stimulates production of an antibody within an organism

Question 5

What is a common biotech tool used in research laboratories?

Answer 5

• Inject a non-human animal (e.g., rabbit or mouse) with a human protein of interest
• The animal will create a specific antibody that will have high affinity for your protein of interest

Question 6

Once purified, from the animal’s serum, how can the antibody be used?

Answer 6

In a variety of immunological techniques to follow your protein of interest including isolation of your protein of interest from cellular lysates by a specialized type of affinity chromatography called immunoprecipitation (IP)

Question 7

What will we isolate in this lab and how?

Answer 7

FLAG-PTEN by using an anti-FLAG antibody attached to a Protein A agarose matrix resin to capture FLAG-tagged PTEN that was expressed in mammalian HEK 293 cells last week

Question 8

What is a major constraint on using IP?

Answer 8

The affinity of the antibody to the antigen (remember that the technique requires the formation of a protein antigen-antibody complex within a cell lysate solution that contains a relatively low concentration of the protein antigen)

Question 9

What antibody affinities are required for quantitative immunoprecipitation?

Answer 9

10^8 mol-1 or higher

Question 10

What happens if the concentration of the protein is too low?

Answer 10

It may not be possible to efficiently isolate the protein antigen quantitatively from the solution by IP

Question 11

What major types of antibody preparations can be used for immunoprecipitation?

Answer 11

Polyclonal and monoclonal antibodies

Question 12

What do polyclonal antibodies contain?

Answer 12

Antibodies that bind to multiple sites on the antigen and therefore often have a higher level of affinity

Question 13

What happens if you capture your antibody-antigen complex on a solid matrix (i.e., protein A)?

Answer 13

The availability of multiple binding sites allows for the formation of a stable antibody-antigen-protein-A complex

Question 14

Although the use of polyclonal antibodies forms a stable multivalent interaction, what does it allow for?

Answer 14

Higher levels of non-specific backgrounds than using a monoclonal antibody

Question 15

Why do polyclonal antibodies have higher levels of non-specific backgrounds?

Answer 15

Partly due to the fact that polyclonal antibodies are isolated from whole sera containing the entire “cocktail” of circulating antibodies that were found within the immunized animal at the time the serum was isolated

Question 16

What can circulating antibodies found within the immunized animal cause?

Answer 16

Contaminating activities and increased non-specific interactions to occur if the polyclonal antibody of interest was not efficiently purified from the rest of the antibodies in the sera

Question 17

Compared to polyclonal antibodies, what do monoclonal antibodies demonstrate?

Answer 17

• A higher level of specificity to their antigen
• Only have the ability to bind to a single epitope (an immunologically active binding site on an antigen to which an antibody or a B or T cell receptor becomes attached), and therefore they provide an excellent tool to assist in identifying a particular structure on an antigen

Question 18

Why else is the level of nonspecific binding greatly reduced?

Answer 18

Since the immune complex that formed between an antigen and a monoclonal antibody typically is not multimeric and is smaller than polyclonal antibodies

Question 19

What is the largest problem with utilizing monoclonal antibodies?

Answer 19

The affinity towards the antigen because the antigen is only connected by the formation of a single antibody-antigen interaction and the bond can easily be disrupted

Question 20

In this lab, how will we be isolating PTEN?

Answer 20

Using a FLAG expression system

Question 21

Our expression plasmid that encodes for PTEN proteins will contain what?

Answer 21

Hydrophilic nature, the FLAG peptide is likely to be located on the surface of the fusion protein and as a result, the FLAG peptide is easily accessible for interaction with an anti-FLAG antibody

Question 22

What does the small size of the FLAG peptide tag not likely to do?

Answer 22

Obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein

Question 23

What FLAG-tag will be used for this lab?

Answer 23

An ANTI-FLAG M2 monoclonal antibody (IgG1) during the IP procedure, that has been purified from a murine cell culture that binds to FLAG fusion proteins