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BB 490 Topic 5E
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Terms in this set (31)
Protein purification meaning
Separating protein of interest from other molecules
Purpose of "bucket biochemistry"
Get natural source proteins via large quantities of starting material (pre-late 1970s)
Recombinant proteins
DNA encoding protein inserted into vector, used by organism (such as E coli) to produce protein (since late 1970s)
Assay for enzymes
Enzyme activity
Western blot assay
Antibody for protein of interest is used to visualize it in a gel
Assays for proteins
Enzyme activity (for enzymes), western blot, check for band at correct position on gel
Which molecules move most slowly through a standard gel?
Largest and least negative molecules
SDS-PAGE detergent
SDS (sodium dodecyl-sulfate)
SDS-PAGE gel material
Polyacrylamide
Function of SDS
Denature protein, shape into rod, and coat with a negative charge
What determines speed of proteins in SDS-PAGE?
Size (and not charge)
Function of reducing SDS gels
Split proteins bound by disulfide bonds into two bands
Reducing agents used in SDS
BME (beta-mercaptoethanol), DTT (dithiothreitol)
Uses of mass spectrometry for protein analysis
Verify, identify, and sequence proteins
Mass spectrometry process for identifying proteins
Volatize protein of interest into vacuum, separate based on mass to charge ratio, get molecular weight information
Mass spectrometry process for sequencing proteins
Cut protein into peptides using proteases, sequence each peptide
Size exclusion (aka gel filtration) chromatography
Gel contains beads with pores that trap and slow small molecules, letting the largest molecules elute first
Ion exchange chromatography
Separates proteins based on charge using a charged column
Cation exchange chromatography
Negatively charged column only lets negatively charged proteins through
Anion exchange chromatography
Positively charged column only lets positively charged proteins through
Affinity chromatography
Receptors are attached to column to bind protein of interest until elution buffer is used
Affinity chromatogram
Nonbinding proteins are washed away first, then elution buffer is used to release protein of interest
Dominant method for purifying proteins
Affinity chromatography
Affinity tag
Sequence added to the N or C terminus of a protein to be recognized in affinity chromatography
How tags are cleaved
Protease cleavage site in vector
IMAC
Immobilized metal affinity chromatography (often used for His tags)
Ways to determine 3D structure of proteins
X-ray crystallography, NMR spectroscopy, CryoEM
X-ray crystallography process
Crystallize pure protein, get defraction pattern after applying x-rays, get electron density map, use electron density to build model of atom locations
Limits of NMR spectroscopy
Proteins must be under 40-50 kDa (400-500 amino acids)
Cryo-electron microscopy process (CryoEM)
Averages thousands of electron micrographs of proteins to get structure
Advantages of CryoEM
Works well for large proteins and complexes, proteins don't need to be crystallized
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