Properties & Calculations Flashcards

Question

What properties are involved to determine the charge and separation of amino acids?

Answer 1

-at pH 2.5 the alpha amino groups exist as NH3+ while carboxylate groups exists as 50% COO- and 50% COOH giving the amino acids an overall positive charge -side chains -exact value of charge depends on specific pKa values of the various groups in each amino acid -size of net charge determines how tightly each amino acid bind -high Na+ in elution buffer displaces weakly bound amino acids and an increase means more tightly bond amino acids

Answer 2

-isolating a specific protein -the cells are broken open to release the proteins into a solution- crude extract -ion exchange is based on charge differences among proteins -depends on the relative number of Asp+Glu (negative versus His+ Lys+Arg (positive) in each protein and on pH -65% of all proteins are negatively charged at pH &

Answer 3

-at different pH look for pro/dep then determine charge -only 5 have charged at pH 7 -given pH 7 is the easiest to calculate charge with -multiple amino acids impact the total net charge -amino group at N-terminal and carboxylate group at c-terminal -anything neutral won't bind -high pH=protonated

Answer 4

-ligand (chemical group) is covalently attached to the beads in the column (inside is stationary phase) -proteins that have an affinity to the ligand bind tightly to it and are put through the column -others elute first and move faster (by addition of high concentration of salt) -atp is isolated

Answer 5

-a peptide or protein that binds a ligand with high affinity and specificity that is fused to the gene encoding target protein -allows purification by affinity chromatography -fuse two genes together one codes for amino acid and one is the fusion gene genes

Answer 6

-clusters of His in a protein bind tightly to Ni2+ or Co+ (high affinity) -6-8 His residues are fused to the target protein at N- or C- terminus (His Tag) -column is made up of chelating resin containing Ni2+ -bound protein is eluted by adding imidazole to the buffer and out competes His-tag and protein no longer binds to the column -high degress of purification in one step

Answer 7

-gel filtration (gel is staionary phase) or molecular exclusion chromatography -beads of polymeric gel with many water-filled stationary pores which proteins can fit through (elute first) -larger proteins are excluded from the pores -small one can enter pores and elute later -use to determine weight of unknown proteins

Answer 8

-measure elution volume of protien of known mass -Vo is the volume of buffer needed to move a protein from top to bottom of coloumn -a linear function of log molar mass -use antilog to determine the molar mass of unknown -find slope (y=mx+b) -in graph: large protein first unknown protein high peak and small protein has the highest volume but smallest peak

Answer 9

-the movement of charged molecules in an electric field -proteins can be separated and characterized using this -used to estimate number of different proteins in a mixture -degree of purity of a mixture -isoelectric point -approximate molecular weight -rate of movement depends on size, shape and charge -go through gel, friction occurs (slower) and shape determines speed -more charge +=faster -larger (-)=slower smaller (-)=faster

Answer 10

-has right porosity for protein 10kDa-1000 kDa -electrophoresis is carried out in gels made up of polyacrylamide and poured between sheets -5-15% polymer 90-95% water with conductive buffer -separated protein are shown by adding a dye (coomassie blue) which binds to proteins

Answer 11

-separation based on isoelectric point: pH at which the net charge on a protein is zero -high pH-deprotonated and moves toward the positive electrode -gradient of decreasing ph becomes protonated and net -ve charge decreases -net charge=0 protein stops moving -pH decreases as you travel down the gel

Answer 12

-separation of complex samples -lots of proteins with similar properties -combines isoelectric focusing and SDS electrophoresis -can separate individual proteins in very complex mixtures

Answer 13

-provides a way to identify proteins -vaporized by laser beam yielding chargd protein particles -larger mass=slower velocity -the time of flight to the detector yield a very accurate mass measurement -compare the mass of the protein with a database of proteins of known mass that allows identification

Answer 14

-catalyze chemical reactions -specific function helps us identify what the protein is -substrate--protein----Product -dont use the protein just isolate it -the amount of target enzyme present after each method can be determined by measuring its activity -moles of substrate converted to produce per unit time

Answer 15

-the total units of enzyme present in a solution -1 enzyme unit is defined as the amount of enzyme that convert 1 umol of substrate to product per minute -should get the same enzyme activity whether the sample is pure or impure

Answer 16

-the number of enzyme unites per milligram of the total protein -enzyme activity/ total protein -purification increases it to reach a max and remians constant once the enzyme is pure

Answer 17

-when pure and impure samples of same enzyme are compared and specific activity is measured by this

Answer 18

-enzyme activity decreases since successive separation methods lead successfully to the loss of some of the protein of interest -total protein decrease (goal is to remove unwanted proteins -specific activity increases (loss of unwanted non-specific proteins is greater than the loss of activity -further purification steps don't lead to an increase in specific activity once protein is successfully isolated

Properties & Calculations Flashcards

(42 cards)