BIOLOGY 2B03 Lecture Notes - Lecture 5: Polyacrylamide Gel Electrophoresis, Sodium Chloride, Gtpase
6 Jun 2018
School
Department
Course
Professor
Biomembranes and Cell Architecture
Why Would a Scientist Purify Proteins?
structural analysis •
functional analysis •
determination of amino acid sequence •
development of antibodies to a protein •
How to Purify Proteins?
1) Develop a good assay
a method of detecting for the presence of a specific protein and estimating the concentration of the protein •
the assay should be specific to the protein you are studying and based upon unique characteristics •
don't mistake another protein for your protein ◦
Ex. enzymatic activity, immunological assay (antibody), biological activity/function •
2) Select protein source
easily obtainable in large amounts (i.e. tissue/ cultured cells) •
contain a high concentration of the protein of interest (POI) •
low in proteins that may co-purify •
low in proteases that may destroy the POI •
3) Break open cells - protein extract
4) Solubilize protein
soluble proteins include: •
cytosolic and secreted proteins ◦
insoluble proteins include: •
transmembrane proteins and membrane-associated proteins ◦
5) Stabilize protein
important parameters in protein stabilization: •
temperature - warm can denature ◦
protease inhibitors - prevents degradation ◦
ligands ◦
salts ◦
metal ions ◦
concentration - can aggregate if at high concentration ◦
pH ◦
6) Fractionate
what criteria can be used to separate POI from other proteins? •
proteins vary in: •
size - separation done via gel electrophoresis or gel filtration chromatography ◦
polarity - adsorption chromatography or hydrophobic interaction chromatography ◦
charge - separation done via ion exchange chromatography or gel electrophoresis ◦
solubility ◦
shape - aka specificity of binding separated by affinity chromatography ◦
Differential Centrifugation: method to begin protein isolation. Separates components in extract based on mass
and density
spin tubes un ultracentrifuge at 1000g to produce pellet at bottom with nuclei and chloroplasts + aq supernatant •
Document Summary
Structural analysis functional analysis determination of amino acid sequence development of antibodies to a protein. Sds-page electrophoresis another way of separating proteins - intentionally denature proteins and coat them results in proteins with negative charge and eliminates the effects of shape and charge density mixture then loaded onto polyacrylamide gel. Lane 2 = fraction obtained after ion exchange chromatography. Lane 3 = fraction after gel exclusion chromatography. Lane 4 + 5 = protein extract after affinity chromatography. Sds-page can be combined with western blot proteins separated by size on gel and transferred to membrane to incubate with solution with antibody to poi and be detected! specific antibodies are used to identify poi: determine purity. Speci c activity = enzyme activity/amount of protein (mg) this determines purity and requires assay. Ex. volume of original cell extract = 1400 ml, total protein present = 10,000 mg, and activity of poi is 100,000 units.
