Amino Acid Analysis Flashcards
1
Q
What is metal affinity chromatorgraphy
A
- clusters of His in a protein bind tightly to Ni2+ or Co2+
- column made up of chelating resin, containing Ni2+
- gene can be modified y adding 6-8 extra HIS residues at N or C term, called HIS-TAG\
- his tag binds tightly to Ni2+ resin - his tag eluded by adding imidazole to buffer
2
Q
What is ultracentrifugation?
A
- sample placed in ultracentrifuge
- at these g forces molecules sediment (move down tube) at rate that depends on size and shape - by measuring the sedimentation velocity, we can calculate the molecular mass of protein
3
Q
- separation based on movement of charged molecules in an electric field
- rate of movement depends on size, charge and shape - carried out through porous gel
- typically gel is 5-15% polymer and 90-95% water
- no modifications are done to protein
A
What is Electrophoresis
4
Q
- sample placed in ultracentrifuge
- at these g forces molecules sediment (move down tube) at rate that depends on size and shape - by measuring the sedimentation velocity, we can calculate the molecular mass of protein
A
What is ultracentrifugation?
5
Q
What is mass spectrometry
A
- identification method not separation method
- protein is vaorpized by laser beam, yielding charged protein particles (pos charge)
- particles travel toward the detector
- velocity depends inversly on the mass (larger=slower)
- The “time of flight” to the detector measures time it takes for molecules to go through tube, measures mass of protein
- we can compare mass of peotein in a database to find what protein it is
6
Q
What is reversed phase chromatography
A
- none-polar hydrocarbin silicon derivative used instead as stationary phase
- polar solvent used as mobile
- used bc better at distingushing subtle differences in hydrocarbon side chains of aa
7
Q
- clusters of His in a protein bind tightly to Ni2+ or Co2+
- column made up of chelating resin, containing Ni2+
- gene can be modified y adding 6-8 extra HIS residues at N or C term, called HIS-TAG\
- his tag binds tightly to Ni2+ resin - his tag eluded by adding imidazole to buffer
A
What is metal affinity chromatorgraphy
8
Q
How can you find the charge of a protein
A
- charge on each aa, add up all aa charges in protein
- look at pKa, charge either pos, neutral, neg or you have to calculate it if it falls within range
9
Q
- sep on basis of molecular size
- largest mol emerge first
- stationary phase= hydrated gel, formed from polymer which absorbs water forming an aqueous network of open pores
- gel is in form of breads/granules. molecules in sample are small enough to fit into pores, since stationary phase these mol progress through column more slowly
- larger mol can’t fit in pores, stay in buffer and go down faster
A
What is gel filtration chromatography
10
Q
How is gel filtration used to estimate molecular mass of proteins
A
- elution volume of proteins is a negative slope linear function of Log molecular mass
11
Q
What is ionic exchange chromatography
A
silica gel replaces by ionic resins
- Cation exchange resins: based on polymers with NEG carboxylate groups, will bind to pos ions/cations
- anion exhange resins: made from polyerswith pos amino groups, bind to neg ions/anions
- solutes now bind according to charge not polarity
- aa can be eluted by adding NaCl to buffer so that Na+ binds in exchange for the pos aa. weakly bound aa displaced @ low NaCl conc, while highly bound require higher conc
- aa detected/ conc measured as they come out column
12
Q
-method to separate components of a mixture - liquid solvent/ buffer flows past particles and is non polar *mobile phase - aa rapidly exchange places -polar aa move much more slowly than non polar aa in solvent
A
What is partition chromatograpy
13
Q
- granular solid (like silica gel) packed into glass tube/column
- buffer containing sample mixture applied to top and then solvent/buffer allowed to flow through to collection tube
- volume buffer needed to move a comound though column is elution volume. Conpounds can be identified by their characteristic elution volume
A
What is column chromatography
14
Q
What is column chromatography
A
- granular solid (like silica gel) packed into glass tube/column
- buffer containing sample mixture applied to top and then solvent/buffer allowed to flow through to collection tube
- volume buffer needed to move a comound though column is elution volume. Conpounds can be identified by their characteristic elution volume
15
Q
How does two-dimensional gels separate complex samples
A
- combines isoelectric focusing and SDS electrophoresis
- lets you isolate based on isoelectric pt and then based on size
