BCHM Exam 1 Answered 100% Correct
Advantage of Absorbance reading - ANSWER--Don't need to do anything to the sample.
Can do the absorbance then use that same thing for another test.
Advantages of affinity chromatography - ANSWER-If isolate ribose binding protein from
cell, may have a whole bunch of other stuff sticking to it (some have high affinity while
others have low or accidental). So when you first load ribose binding on to the column,
you can do a wash step with NaCl. This takes those interactions not mediated by
hydrophobic interacts like tertiary or Quaternary, it provides enough distraction for those
with low affinity for it to fall off. At this point you would then add in your free ligand which
will then cause the functional complex to fall off.
Affinity - ANSWER-A protein may have many different ligands while a ligand can also
interact with many different proteins. The preference of a protein for a ligand is its
affinity for that ligand.
Affinity chromatography - ANSWER-Taking advantage of protein ligand interaction.
So if want to purify ribose binding protein, your column would have ribose on it. So the
ribose binding protein will stick. You can then elute them off the column by adding free
ribose, or can allute by changing the pH. This will impact the proteins ability to bind to
the ribose in the first place.
Allostery - ANSWER--When protein conformational changes affect protein function
-conformation change affects protein/enzyme function
Antigens - ANSWER-All antigens have the same common core that is common for that
SPECIES
Aspect of salt bridge formation - ANSWER-Strong positive and negative charge
between two residues that are not close in primary structure but close in tertiary so they
are 3D close to each other that forms a ionic bond between them that is a significant
reaction that has these subunits have more interactions making it harder to leave the T-
state cause that would require more bonds to be broken.
Beta Sheet - ANSWER--Amino acids that are in a semi-extended form, making rows
that form a (pleated) plane.
-The rows in a sheet may be parallel or anti-parallel
- There are H bonds between rows
-The side chains are perpendicular to the surface of the sheet
Binding pocket - ANSWER-Part of protein specifically interacting with ligand
,Bradford Assay - ANSWER-Depends on dye. When not bound to protein would be red,
and when bound to protein would be blue. Using standard curve again can find out total
protein quantification.
Can either T and R state have oxygen - ANSWER-Either one can be with or without
oxygen but T better without and R better with
Can you find parallel and anti parallel sheets together - ANSWER-yes, it doesn't have to
be one or the other
Cooperativity (Hill coefficient) - ANSWER-Ligand binding at one site affects ligand
binding at another site.
Describe absorbance - ANSWER-If we assume an even distributions of Tyr, and trp.
This absorbance (using a standard curve) you would be able to determine mass of
protein in the sample
Describe Anti parallel Beta sheet - ANSWER--Backbone within plan of sheet
-The side chains alternate coming in then our of the board
Describe Isoelectric Focusing - ANSWER-Another version of PAGE without the SDS
-This sorts things on PI rather than MW
-So set up polyacrylamie gel with a pH gradient
-Then load things on one side and pass current through gel.
-The proteins move through gel until they become neutral (reach their PI)
-Then stain with coomassie to see
Describe parallel beta sheets - ANSWER-Side chains alternate coming in then our of
the board
Describe the anion exchange - ANSWER-There will be a cylinder with a bunch of
positively charged beads. You then pour the beaker with your stuff in it over the column
and collect whats at the bottom. Those with positive charge will flow right through.Those
with some level of charge will stick. Then you change flow through tube (this new one is
now fraction 1). Then you add 50 mM NaCl. NaCl has positive (Na+) and negative parts
(Cl-).So the few Cl- will bind to the column and bump off those with low negative charge
so those guys go to the flow through. The Na+ goes by and the low negative charge
sees them and tries to attach to them instead of the column which is why they go to the
flow through.
Now you increase salt concentration to 150mM NaCl. So this is where the middle
negative charge goes into fraction 2. then add 0.5M NaCl which finally causes those
with high negative charge to flow through into fraction 3.
So depending on which one we want (like if we wanted the positive charge, we would
do the first step since this stuff is now in the first flow through, then clean the column)
can you just do whatever order you want.
, Describe the loops ( and turns and coils) - ANSWER-- This usually happens on the
surface of proteins and have hydrophilic residues
1.) Turns- are 3 to 5 residues that has an internal H bond
2.)Random coil- are long loops that have no determined structure
Describe the peptide bond - ANSWER-Two amino acids together with the connection
between them being a N and H with two electrons free
Difference between coomasie blue and western blot - ANSWER-Coomasie shows
where all the protein are, western blot only detects protein you target
Diisulfide bonds - ANSWER-between 2 beta sheets or between beta sheets and alpha
helices. They are close together in 3D shape not primary structure
dimer and trimer - ANSWER--Two different proteins coming together
-Three proteins coming together
Disadvantage of Bradford assay - ANSWER-Disadvantage:
-Can't use sample after dye is added.
Advantage:
-Much more sensitive
Disadvantages of endogenous - ANSWER--Can't study specific protein from human
-Difficult if protein of interest isn't made in high quantities naturally
Do big proteins move fast or slow through the SDS page. - ANSWER-Small proteins
move through the gel faster while big ones go slowly
Do we usually do exogenous or endogenous expression - ANSWER-Exogenous
Do you use Coomasie blue and western blot together or seperate? - ANSWER-Use
coomasie blue and western blot in tandem. So do two different gels, and do one with
coomasie and one with western blot
Domain - ANSWER-Independently folding piece of tertiary structure
For a polyprotic acid each H+ has same or different pKa - ANSWER-different which is
why we can treat independently if far enough apart
Fraction occupancy - ANSWER-Fraction of binding pockets occupied by ligand
theta=[P:L]/[Ptot]=[P:L]/[P]+[P+L]
Advantage of Absorbance reading - ANSWER--Don't need to do anything to the sample.
Can do the absorbance then use that same thing for another test.
Advantages of affinity chromatography - ANSWER-If isolate ribose binding protein from
cell, may have a whole bunch of other stuff sticking to it (some have high affinity while
others have low or accidental). So when you first load ribose binding on to the column,
you can do a wash step with NaCl. This takes those interactions not mediated by
hydrophobic interacts like tertiary or Quaternary, it provides enough distraction for those
with low affinity for it to fall off. At this point you would then add in your free ligand which
will then cause the functional complex to fall off.
Affinity - ANSWER-A protein may have many different ligands while a ligand can also
interact with many different proteins. The preference of a protein for a ligand is its
affinity for that ligand.
Affinity chromatography - ANSWER-Taking advantage of protein ligand interaction.
So if want to purify ribose binding protein, your column would have ribose on it. So the
ribose binding protein will stick. You can then elute them off the column by adding free
ribose, or can allute by changing the pH. This will impact the proteins ability to bind to
the ribose in the first place.
Allostery - ANSWER--When protein conformational changes affect protein function
-conformation change affects protein/enzyme function
Antigens - ANSWER-All antigens have the same common core that is common for that
SPECIES
Aspect of salt bridge formation - ANSWER-Strong positive and negative charge
between two residues that are not close in primary structure but close in tertiary so they
are 3D close to each other that forms a ionic bond between them that is a significant
reaction that has these subunits have more interactions making it harder to leave the T-
state cause that would require more bonds to be broken.
Beta Sheet - ANSWER--Amino acids that are in a semi-extended form, making rows
that form a (pleated) plane.
-The rows in a sheet may be parallel or anti-parallel
- There are H bonds between rows
-The side chains are perpendicular to the surface of the sheet
Binding pocket - ANSWER-Part of protein specifically interacting with ligand
,Bradford Assay - ANSWER-Depends on dye. When not bound to protein would be red,
and when bound to protein would be blue. Using standard curve again can find out total
protein quantification.
Can either T and R state have oxygen - ANSWER-Either one can be with or without
oxygen but T better without and R better with
Can you find parallel and anti parallel sheets together - ANSWER-yes, it doesn't have to
be one or the other
Cooperativity (Hill coefficient) - ANSWER-Ligand binding at one site affects ligand
binding at another site.
Describe absorbance - ANSWER-If we assume an even distributions of Tyr, and trp.
This absorbance (using a standard curve) you would be able to determine mass of
protein in the sample
Describe Anti parallel Beta sheet - ANSWER--Backbone within plan of sheet
-The side chains alternate coming in then our of the board
Describe Isoelectric Focusing - ANSWER-Another version of PAGE without the SDS
-This sorts things on PI rather than MW
-So set up polyacrylamie gel with a pH gradient
-Then load things on one side and pass current through gel.
-The proteins move through gel until they become neutral (reach their PI)
-Then stain with coomassie to see
Describe parallel beta sheets - ANSWER-Side chains alternate coming in then our of
the board
Describe the anion exchange - ANSWER-There will be a cylinder with a bunch of
positively charged beads. You then pour the beaker with your stuff in it over the column
and collect whats at the bottom. Those with positive charge will flow right through.Those
with some level of charge will stick. Then you change flow through tube (this new one is
now fraction 1). Then you add 50 mM NaCl. NaCl has positive (Na+) and negative parts
(Cl-).So the few Cl- will bind to the column and bump off those with low negative charge
so those guys go to the flow through. The Na+ goes by and the low negative charge
sees them and tries to attach to them instead of the column which is why they go to the
flow through.
Now you increase salt concentration to 150mM NaCl. So this is where the middle
negative charge goes into fraction 2. then add 0.5M NaCl which finally causes those
with high negative charge to flow through into fraction 3.
So depending on which one we want (like if we wanted the positive charge, we would
do the first step since this stuff is now in the first flow through, then clean the column)
can you just do whatever order you want.
, Describe the loops ( and turns and coils) - ANSWER-- This usually happens on the
surface of proteins and have hydrophilic residues
1.) Turns- are 3 to 5 residues that has an internal H bond
2.)Random coil- are long loops that have no determined structure
Describe the peptide bond - ANSWER-Two amino acids together with the connection
between them being a N and H with two electrons free
Difference between coomasie blue and western blot - ANSWER-Coomasie shows
where all the protein are, western blot only detects protein you target
Diisulfide bonds - ANSWER-between 2 beta sheets or between beta sheets and alpha
helices. They are close together in 3D shape not primary structure
dimer and trimer - ANSWER--Two different proteins coming together
-Three proteins coming together
Disadvantage of Bradford assay - ANSWER-Disadvantage:
-Can't use sample after dye is added.
Advantage:
-Much more sensitive
Disadvantages of endogenous - ANSWER--Can't study specific protein from human
-Difficult if protein of interest isn't made in high quantities naturally
Do big proteins move fast or slow through the SDS page. - ANSWER-Small proteins
move through the gel faster while big ones go slowly
Do we usually do exogenous or endogenous expression - ANSWER-Exogenous
Do you use Coomasie blue and western blot together or seperate? - ANSWER-Use
coomasie blue and western blot in tandem. So do two different gels, and do one with
coomasie and one with western blot
Domain - ANSWER-Independently folding piece of tertiary structure
For a polyprotic acid each H+ has same or different pKa - ANSWER-different which is
why we can treat independently if far enough apart
Fraction occupancy - ANSWER-Fraction of binding pockets occupied by ligand
theta=[P:L]/[Ptot]=[P:L]/[P]+[P+L]
