Protein Purification Flashcards
T/F all functional proteins require at least 2 polypeptide chains
FALSE. some are functional with just one chain
name of proteins based on number of chains
dimer: 2 chains
trimer: 3 trains
tetromer: 4 chains
what characteristics are proteins separated by
size
charge
unit for size of proteins
dalton
steps for protein purification
-isolate protein
-detect protein
-assay protein activity
-separation technique
-quantitation
methods to detect proteins
UV
Bradford Assay
UV protein detection
-works for aromatics
-absorbance at 260-280 nm
-not very sensitive(50-100 ug needed per mL)
Bradford Assay protein detection
-uses coomasie blue as binding dye
-absorbance at 595 nm
highly sensitive(1 ug of protein per mL)
what is the protein extinction coefficient
0.02 mL mg-1 cm-1
Beer-Lambert Law
A = εlc
A: absorbance
ε: extinction coefficient
I: pathlength(usually 1 cm)
c: concentration
protein purification techniques
-salting out(solubility)
-ion exchange chromatography(charge)
-hydrophobic interaction chromatography(polarity)
-gel filtration chromatography(size)
-affinity chromatography(binding affinity)
salting out method
-start with low salt concentration to precipitate unwanted proteins
-use higher salt concentration to precipitate target protein
ion exchange method
cation exchange-column is negatively charged and protein is positively charged
anion exchange-column is positively charged and protein is negatively charged
opposite charges bond to keep desired protein
hydrophobic interactions method
-add high salt concentration to help non-polar groups on protein bind with the phenyl group
-as salt concentrations lower hydrophobic proteins elute(come off)
-the most hydrophobic proteins come off last
gel filtration method
-proteins are run through carbohydrate polymer beads containing small tunnels
-large molecules travel through quicker since they dont travel through the tunnels
-takes time, so only good for stable proteins
Affinity chromatography method
-resin contains beads with a certain chemical group with binding affinity for protein
-desired protein binds while non desired travels
-glucose added and proteins are released
what is the trend of fraction volume, total protein, and specific activity as separation techniques progress
volume and total protein:decrease
specific activity: increase
what is the trend of the specific activity(ratio) as separation techniques progress
increase
gel electrophoresis
SDS detergent used to give all proteins a negative charge(separate on size not charge)
-voltage pushes proteins through gel, highest MW travels the least, lowest MW travels most
isoelectric focusing(1D)
-determines pI of protein
-pH gradient is established in gel
-protein is added and moves until it reaches its isoelectric point
2D isoelectric focusing
-apply 1D gel to SDS gel
-separated according to molecular weight
