Protein Quantification Flashcards
What are the three steps of protein quantification?
- Separate/purify
- Identify
- Quantify
What are four types of chromatography?
HPLC, Affinity, Size exclusion, and Ion exchange
What are the three detectors for proteins?
- UV
- Fluorescence
- Refractive Index
Where is the main action of chromatography occuring?
On the column
What is a way that a chromatography column can separate materials?
hydrophilicity
Explain how materials will come out in a chromatography column based on their hydrophilicity.
More hydrophilic materials will come out of the column first because of their high affinity for the mobile phase. Then the more hydrophobic materials will come out later
Intensity of protein absorption signal is (disproportionate or proportionate to concentration.
proportionate
Retention time is affected by ______ and ____.
Retention time is affected by the column and solvent.
Explain functional groups and how they come out of the column.
The longer chains they have, the more hydrophobic, and the quicker they come out of the column
Color is ___ to concentration.
proportional
Explain how ELISA works?
A plate that has a lot of capture antibodies and your protein will attach to the antibody. then you add a secondary biotin antibody that will interact with your protein. Then add an enzyme and substrate that will produce a color. If you observe a color, you know your protein is there.
What is biotin streptevadin?
A large protein with dimers
What is biotin commonly used for?
conjugation
What is an advantage of ELISA?
It is very sensitive and you can detect very small amount of protein
What is important to know before you choose a quantification method?
An approximate concentration range because each method has an accuracy range
What are the advantages of chromatography?
easy, multiple columns and solvents available, very sensitive to small amounts of proteins
What are disadvantages to chromatography?
Only be used for proteins in solution (cant use for proteins on the surface
What are the advantages for colorimetric and fluorescent assays?
surface proteins/solution, easy to get quantitative data
What are the disadvantages for colorimetric and fluorescent assays?
many require pre-labeling
What are the advantages for ELISA?
very sensitive to small amounts (pg), can also use on the surface and solution
What are the disadvantages for ELISA?
requires antibodies to interact with the protein target, expensive
What are the advantages for the western blot?
Can be used to probe multiple proteins in the same sample
What are the disadvantages for the western blot?
requires antibodies to protein target, proteins must be in solution, less sensitive than ELISA
