Amino Acid Analysis Flashcards
What is metal affinity chromatorgraphy
- 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
What is ultracentrifugation?
- 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
- 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
What is Electrophoresis
- 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
What is ultracentrifugation?
What is mass spectrometry
- 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
What is reversed phase chromatography
- 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
- 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
What is metal affinity chromatorgraphy
How can you find the charge of a protein
- 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
- 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
What is gel filtration chromatography
How is gel filtration used to estimate molecular mass of proteins
- elution volume of proteins is a negative slope linear function of Log molecular mass
What is ionic exchange chromatography
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
-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
What is partition chromatograpy
- 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
What is column chromatography
What is column chromatography
- 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
How does two-dimensional gels separate complex samples
- combines isoelectric focusing and SDS electrophoresis
- lets you isolate based on isoelectric pt and then based on size
What is partition chromatograpy
-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
What is gel filtration chromatography
- 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
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
What is ionic exchange chromatography
-silica gel spread thinly - samples applied to gel -lower edge dipped in solvent aa can be identified by relative mobility -most polar @ bottom
What is thin layer chromatography
- separation based on isoelectric point of proteins
- isoelectric pt= pH at which net charge on protein is 0
- at high pH, protein deprotonated moved towards the + electrode -as it passes through gradient of decreasing pH, it becomes protonated, net negative charge decreases
- when net charge=0, protein stops moving
- become separated along the pH gradient
What is isoelectric focussing?
What is relative mobility
- in thin layer chromatgraphy, how far amino acid moves
- very polar aa have low Rf anf non polar have high Rf
- calculated by Rf=Xb/Xf (Xb = how far moved, Xf = distance to solvent front)
What is isoelectric focussing?
- separation based on isoelectric point of proteins
- isoelectric pt= pH at which net charge on protein is 0
- at high pH, protein deprotonated moved towards the + electrode -as it passes through gradient of decreasing pH, it becomes protonated, net negative charge decreases
- when net charge=0, protein stops moving
- become separated along the pH gradient
What properties of proteins are the basis of separation using SDS-PAGE
- size of protein
What is the use of sodium dodecyl sulphate (SDS)
- SDS causes protein molecules to extend (linearize) and gives a uniform charge per unit size
- native protein charge is “swamped out”, and overall negative charge is used to move the protein through the gel towards pos electrode ** shape and charge does not have influence**
- separation is based strictly on their SIZE - small proteins move rapidly down gel while large “meander”
- SDS-PAGE may also be used to measure molecular mass of a polypeptide by comparing STANDARDS of known size ** if there are multiple subunits in a protein they usually separate with this method**
What is thin layer chromatography
-silica gel spread thinly - samples applied to gel -lower edge dipped in solvent aa can be identified by relative mobility -most polar @ bottom
What is Electrophoresis
- 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
- 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
What is reversed phase chromatography
