Proteins: Determination of Primary Structure and Expression Flashcards
What are seven types of proteins with different functions?
Enzymes, hormones, transport, storage, structural, protective, contractile
What is the difference between a simple protein and a conjugated protein?
Simple protein is compose solely of amino acids while a conjugated protein has non AA components.
What are examples of conjugated proteins?
lipoproteins, glycoproteins, phosphoproteins, hemoproteins, flavoproteins, metalloproteins
How many amino acids are required for a protein to fold?
40
What is the common number of AA in proteins?
100-1000
How many genes in humans? How many expressed?
40,000 and 10k-15k
Some proteins are expressed at high levels and others at low. Give an example of each.
High: collagen
Low: hormones
Why are hormones harder to research than collagen?
Low expression makes identification difficult. Rely on animal hormones for research
What characteristics can be used to separate proteins?
solubility, charge, size, and affinity
Technique; Match to Characteristic
Ion Exchange Chromatography
Isoelectric focusing
Electrophoresis
Charge
Technique; Match to Characteristic
Dialysis and ultracentrifugation
Gel electrophoresis
Gel filtration
Size
Technique; Match to Characteristic
2D gel electrophoresis
Charge and size
Technique; Match to Characteristic
Affinity Chromatography
Specificity
Technique; Match to Characteristic
Paper Chromatography
Reverse-Phase Chromatography
Hydrophobic Chromatography
Polarity
In affinity chromatography, the _____ attaches to the stationary phase which is the ligand. This occurs in a lot of test tubes.
target
Why does SDS Page use sulfur?
Mask the charge of the molecules so that they all become negative. Allows samples to move toward positive charge based on MW alone. Speed at which they move toward positive indicates weight.
Western Blot (immunoblotting) occurs after what size filtration technique?
SDS Page or any size technique
In Western Blot, the gel samples are incubated with ______ _____ which are formulated to attach to the specific protein of interest. Then, labeled ______ _____ are added to attach to the _____ ______.
primary antibodies
secondary antibodies
primary antibodies
After the luminescent antibody binds, a colorimetry test can be used to detect the protein of interest. What is a colorimetry test?
A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light.
Does ELISA measure protein expression or activity?
Expression
What are the steps to completing an ELISA?
- Coat surface with antigen.
- Block unoccupied sites with DUMB protein
- Add antibody for antigen
- Add antibody for first antibody. This second antibody has an enzyme on it
- Add substrate for enzyme
- Test degree of color to see degree of enzyme expression
The primary structure of insulin was first determined by ______.
Sanger
What are the steps of insulin maturation?
- Preproinsulin (A, B, C chains)
- Proinsulin (cleavage of signal peptide of N term and disulfide bond formation)
- Insulin (cleavage of C)
Edman Degradation uses ______ cleaving agents to create _______ amino acid sequence.
multiple, overlapping
N terminal amino acids are removed one at a time by binding to _________ or a derivative _____. This is a form of sequencing via ____ _____.
phenylisothiocyanate (Edman’s reagent)
PTH
Edman Degradation
What are two forms of mass spec that can be used to identify proteins?
MALDI and ESI
Mutations in what form of insulin cause neonatal diabetes?
Preproinsulin
What are the benefits of using mass spec for protein identification?
High specificity even if protein isn’t completely pure
Sensitivity allows for quantitative analysis
Can identify full peptides
Samples can be run in parallel for multiple proteins
Can determine post translational modifications with locations
