FINAL EXAM CHAPTER 5 Protein methods Flashcards
How can we test for the presence of a protein?
They have to be purified and this happens on the basis of differences in their chemical properties and this requires a test or assay.
What is specific activity ?
The ratio of enzyme activity to protein concentration. and specific activity should increase with each step of the purification procedure
How does dialysis work?
The protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse but big enough to allow the salt to equilibrate with the surrounding the dialysis bag .
How does gel filtration work?
This is the separation of proteins on the basis of size. A column is filled with porous beads. When a protein solution is passed over the beads, large proteins cannot enter the beads and exit the column first. Small proteins can enter the beads and thus have a longer path and exit the column last.
How does Ion exchange work?
This is the separation of proteins on the basis of charge. The beads in the column are made so as to have a charge. When a mixture of proteins is passed through the column , proteins with the same charge as on the column will exit the column quickly. proteins with opposite charge will bind to the beads and are released by increasing salt concentration or adjusting the pH of the buffer that is passing through the column
How does the affinity chromatography work?
This is done by picking some proteins that have high affinity for specific chemicals or chemical groups. Beads are made with specific chemical attached. A proteins mixture is passed through the column. Only protein with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical to which the protein is bound.
What is Gel Electrophoresis(SDS-Page) ?
This is when migration occurs in a gel and proteins will migrate in an electrical field because they are charged.
How does Gel Electrophoresis(SDS-Page) work?
-Unfolds all the proteins
-reducing agent breaks the all the bonds then they are denatured
-Since they are extended now we can separate things according to their molecular weight
-Smaller molecules will run through the gel
How does the two dimensional gel electrophoresis work?
Proteins area separated in one direction by isoelectric focusing. This gel is then attached to an SDS-Page gel, and electrophoresis is performed at a 90 degree angle to the direction of the isoelectric focusing separation.
What is immunoprecipitation and how does it work?
This is a method that enables the purification of a protein.
-This is when the antigen binds to an antibody and everything else is discarded throughout the process and at the very end they both separate and antibodies are discarded and we are left with protein.
What does Enzyme linked immunosorbent assay do? and how many types are there?
-It quantifies the amount of protein present.
-Indirect ElISA
-Sandwich ELISA
How does indriect ELISA work?
-Starts with antigen coated well
-Specific antibody is added and binds to antigen
-Enzyme linked antibody binds to specific antibody
-Substrate is added and converted by enzyme into colored product; the extent of color formation is proportional to the amount of specific antibody
How does Sandwich ELISA work?
-Starts with a Monoclonal antibody coated well
-Antigen is added that binds to the antibody
-A second monoclonal antibody, linked to enzyme, binds to immobilized antigen
- Substrate is added and converted by enzyme into colored product; the extent of color formation is proportional to the amount of antigen
How does Western Blot work?
Proteins separated in an SDS-PAGE gel , transferred to a sheet of polymer, and then stained with a fluorescent antibody
What does Edman Degradation determine?
Amino acid sequence
