Biotech Final - Lab 8 Flashcards
Immunoprecipitation followed by SDS-PAGE and immunoblotting can be used to?
1) Determine the molecular weight of protein antigens
2) Study protein-protein interactions
3) Determine specific enzymatic activity
4) Monitor protein PTM
5) To determine the presence and quantity of proteins
a) Enables the detection of low abundant proteins
What is an antigen?
A foreign substance that stimulates production of an antibody within an organism
How does one make an antibody to a protein?
1) Inject non-human animal with a human protein of interest
2) Animal will create a specific antibody that will have high affinity for the protein of interest
3) Purify from animal’s serum
In this lab we did what?
Precipitate FLAG.PTEN using mouse anti-FLAG antibody conjugated to protein A agarose resin to capture FLAG tagged PTEN that was ectopically expressed in mammalian Hek 293 cells.
What are the constraints on IP?
1) Affinity of the antibody to the antigen
a) Quantitative IP requires antibody affinities of 10^8 mol^-1 or higher
If the concentration of the protein is low, the protein antigen may be detected by ___________ the lysate but, will not be possible to _____________ the protein antigen quantitatively from solution using ___________.
1) Western blotting
2) precipitate
3) IP
What are the two types of antibody preparations?
Polyclonal and monoclonal
What are the basic steps of IP?
1) Lyse cells and prepare sample for IP
2) Pre-clear sample by running over beads alone (i.e. without antibody, removes contaminants)
3) Incubate solution with antibody against protein of interest (antibody attached before this step)
4) Precipitate complex (centrifugation if using beads, remove supernatant)
5) Resuspend and wash (spin each time)
6) Elute (using laemlli sample buffer with BME)
7) Analyzed complexes or antigens of interest
a) SDS-PAGE
b) MS
c) Western Blot
What is the most common antibody preparation?
Polyclonal
What are polyclonal antibodies?
Antibodies that bind multiple sites on the antigen, have higher affinity but lower specificity. Polyclonal antibodies are isolated from whole sera and thus contain more contaminants.
What are monoclonal antibodies?
1) Demonstrate a higher level of specificity to their antigen
2) Only bind to one epitope region
3) The level of non-specific binding is reduced
4) The affinity towards the antigen is weaker and can be easily disrupted
Describe the FLAG tag.
1) Short hydrophilic amino acid (sure to be located on surface of fusion protein)
2) 8 aa
3) due to small size, not likely to obstruct protein folding, compartmentalization, secretion, alter functions, block epitopes or hinder protein transport.
What antibody did we use?
anti-FLAG M2 monoclonal antibody (IgG1) purified from a murine cell culture
