4. protein experimental techniques Flashcards
what must researchers do before they can determine: 1. which proteins=expressed in a given cell 2. quantity of specific protein in a cell 3. amino acid sequence of a protein 4. molecular weight of pI of a protein
before they can determine any of these, they must first isolate and purify their protein of interest from thousands of proteins in a cell
to purify a protein, what do you start with
start with a tissue sample or a large collection of bacterial cells
what is the first step of protein purification
they need to make a crude extract
how are crude extracts made
by breaking open the cells you start with via differential centrifugation
what is differential centrifugation
centrifuging your sample at higher speeds each time, which pellets out different cell components after each spin. The product is an impure sample, but all the proteins are of the same type (ie all mitochondrial proteins)
after differential centrifugation (which isolates proteins based on type), what is the next step
the crude extract then undergoes fractionation treatments
what is the purpose of fractionation treatments of the crude extract
this separates the proteins into different fractions based on a specific property (ie size, net charge, binding affinities, solubility, etc)
what is the result of fractionation of the crude extract
a solution with just a few proteins
what is salting out
isolation of a protein based on solubility
what is used in salting out
ammonium sulfate
describe the salting out process
you add salt to precipitate out your proteins. More salt=less soluble proteins become in the solution.
- additional salt disrupts the bonds between water and protein, making the proteins aggregate
- aggregates can be removed via low speed centrifugation
what bonds are disrupted during salting out? what is the result of this
bonds between water and protein, which makes the proteins aggregate
what process usually follows salting out
dialysis
what is dialysis
a process that removes salts and other small molecules from the solution
describe the process of dialysis
the precipitate containing the protein of interest is put into a semipermeable tube and resuspended into a buffer. Only small molecules leave the tubing, leaving you with little/no salt left
what is the purpose of column chromatography
separation of proteins by their properties
name the two phases of column chromatography
the stationary and mobile phase
describe the stationary phase of column chromatography
it is a solid, porous material (ie beads, gel, resin) inside the column, and it has very specific properties
describe the mobile phase of column chromatography
this is your aqueous protein sample
describe the process of column chromatography
a solution from a reservoir is constantly flowing over the column and will eventually drip out the bottom of effluent. The sample is placed on top, proteins migrate down
describe protein migration in column chromatography
proteins in solution go from the top to bottom of the tube. As they interact with the porous matrix, they’ll be slowed to different degrees based on protein properties
describe the end result of column chromatography
you can collect the (now separated) proteins as they exit the column into fractions
what are the consequences of having a bigger column for column chromatography
bigger=better separation
bigger=more diffuse protein gets (not as favourable)
at the end of column chromatography, how do you know if your fractions have protein in them
see if they can absorb light at 280nm, because proteins have some absorption due to aromatic residues
T or F: column chromatography helps you identify which proteins you have in your sample
false; it only helps you isolate proteins based on their properties
what is the purpose of ion exchange chromatography
isolation of proteins based on their net electric charge (sign and magnitude)
describe the process of ion exchange chromatography
get a column with beads that are either neg or pos charged. Run the sample through, and oppositely charged proteins will bind to the beads, similarly charged proteins will flow through
define cation exchangers
these are positively charged proteins that bind to negative beads
define anion exchangers
these are negatively charged proteins that bind to positive beads
what is the purpose of size exclusion chromatography
separate proteins based on size
what is another name for size exclusion chromatography
gel filtration chromatography
why is gel filtration chromatography another name for size exclusion chromatography
because the stationary phase is often a porous gel
what is the result of size exclusion chromatography
larger proteins will elute first, as they can’t fit into the cavities of the gel. Smaller proteins get stuck in the cavities, so it takes them longer to elute out
T or F: smaller proteins elute out more quickly than larger proteins in size exclusion chromatography
false; the smaller ones get stuck in cavities of the gel, so it takes them longer
what is the purpose of affinity chromatography
separation of proteins due to ligand binding affinity
what are examples of ligands that may be used in affinity chromatography
ATP or cAMP
describe the process of affinity chromatography
the beads have a ligand covalently attached, that the protein of interest will bind to. After one pass, unwanted proteins are eluted out, but protein of interest will be stuck to the beads. A wash with a free ligand elutes the desired protein (because free ligand competes with the bound ligand, so protein will bind to free ligand instead and move its way down)
how has column chromatography become more high tech
- high pressure used to push mobile phase through the column instead of waiting for it to drip through
- automated injectors to place mobile phase
- automated/highly sensitive detectors for each released fraction (tells you if/what protein came out)
- entire process is controllable by computer programs
to purify a protein for the first time, what should you keep in mind
lots of trial and error is required. You typically use the cheap methods first, and only the expensive ones when you have a small volume to deal with
what are 2 useful parameters when talking about enzymes
activity and specific activity
define activity
total units of enzyme in the solution
define specific activity
the number of enzyme units per mg of total protein (ie proportion of protein of interest vs all other protein in the sample)
when do you stop the purification process
- when further steps no longer increase the specific activity (only applicable if protein=enzyme)
- stop when only a single protein species is detectable on a gel
what method is used to visualize proteins as you purify
electrophoresis
as you purify, what can electrophoresis help you determine
critical protein properties such as isoelectric point and molecular weight (by the migration in an electric field)
T or F: electrophoresis helps purify proteins
false; it is only used as an analytical method. It does not contribute to purification
what type of gel is used in electrophoresis
polyacrylamide gel
describe polyacrylamide gel
porous, will slow proteins to varying degrees based on their charge/mass ratio and their shape
what is SDS (include full name)
sodium dodecyl sulfate (a detergent)
why is SDS added in the electrophoresis procedure
it will separate a mixture of proteins. Nearly one SDS molecule will bind per amino acid
what type of charge does SDS contribute to
contributes a large negative charge to the protein
T or F: SDS helps denature proteins
true
when SDS is applied to a protein, what is the only property we must consider when looking at the final gel? why
size. With SDS, all will be negative, and all will be unfolded, so size is the only remaining property that could differ between the proteins
T or F: smaller proteins migrate faster through gel
true
what dye is used to stain the proteins after electrophoresis
Coomassie blue
what is the full name for SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
mobility of most polypeptide chains in linearly proportional to ____
the log of their mass
what can SDS NOT do
break disulfide bonds
what is the result of SDS not being able to break disulfide bonds
it can be hard to determine the total number of polypeptides present
name 2 chemicals that can be added to a sample to break disulfide bonds, and thus separate any polypeptides bound by cysteine residues
beta-mercaptoethanol
dithiothreitol
what process is used to determine the isoelectric point of a protein
isoelectric focusing
is SDS used in isoelectric focusing?
no
what is the charge at pI
net charge of 0
describe the process of isoelectric focusing
first we take molecules that either act as acids or bases, and we run them through the gel. Their migration establishes a pH gradient, and then we can run our protein through
in isoelectric focusing, when would protein migration stop
migration stops when pI is reached (net charge of zero)
if a protein is in a region of the gel where the pH is higher than its pI, what charge will the protein have
when pH is higher than pI, we have more deprotonation of functional groups, so the net charge is negative
what is the purpose of 2D electrophoresis
allows you to find a protein’s molecular weight and pI at the same time
what is the first dimension of 2D electrophoresis
isoelectric focusing
what is the second dimension of 2D electrophoresis
SDS-PAGE
describe the process of 2D electrophoresis
use isoelectric focusing, leaving you with a column gel with bands at different pH values depending on their pI values. Place this on SDS polyacrylamide gel, and then run the gel
how do you interpret the results of 2D electrophoresis
the first dimension shows you decreasing pI, while the gel shows you decreasing weight (think of it like 2 axes on a graph)
what are 3 things you can determine with electrophoresis
- how pure your sample is
- protein’s molecular weight
- protein’s isoelectric point
what is the crude way you can use electrophoresis to purify
run the gel, then physically cut the band you want out of the gel and discard the rest
what is the full name of ELISA
enzyme-linked immunosorbent assay
what is ELISA used for
used to determine whether a protein is present in a mixture of proteins
describe the process of ELISA
- coat the surface of the wells with sample (antigen)
- block unoccupied sites with nonspecific protein
- incubate with primary antibody against the specific antigen
- incubate with secondary antibody
- formation of coloured product indicates presence of specific antigen
T or F: in an ELISA, all colored products will be the same intensity
false; the color intensity is proportional to the concentration of the protein of interest in the sample
what is mass spectrometry
a non-gel based method to measure protein molecular weight, or the change in molecular weight when a cofactor or ligand is bound
what does mass spec measure
the time it takes of a protein to travel through an electric field from an injection point to a detector
what are the 3 mass spec methods
- MALDI MS
- ESI MS
- Tandem MS
when is it easiest to determine the amino acid sequence of a protein
when you know the DNA sequence of the protein
how do you determine the amino acid sequence of a protein if the DNA sequence is unknown
the purified protein will be sequenced, and a key part of this will be breaking the polypeptide into smaller fragments
what is one method of breaking polypeptides into fragments when trying to determine the amino acid sequence
edman degradation
what is edman degradation
a method that cleaves off the N-terminal residue, but leaves all other bonds intact. The identity of that residue can be determined, and the process can be repeated for each next amino acid in line
what is the name of the chemical in edman degradation that labels the amino-terminal residue
phenylisothiocyanate (PITC)
how does edman degradation link to pH
in edman degredation, each step alternates between acidic and basic pH
what is one method used to break peptide bonds in an isolated and purified protein
acid hydrolysis
what is the purpose of acid hydrolysis
break peptide bonds from an isolated and purified protein
after acid hydrolysis, what step(s) can be done (and why)
chromatography or spectroscopy can be used to identify what amino acids are present and in what proportions
what is the purpose of proteases
if you know what amino acids are present in a protein but don’t know the order, you can use proteases to digest the polypeptide into a few fragments
how do proteases work
they only cleave the N or C terminal to certain amino acids (not the middle)
after using proteases, how can you determine the amino acid sequence
you can look at where the fragments overlap to determine the order
describe the process of Tandem MS
hydrolyze a protein w/ protease. Injection into the first MS where proteins separate based on mass + charge. One species is selected to go into a collision cell, where it is broken down a bunch more times at random peptide bonds. Run the second MS
what is the result of a Tandem mass spec
you get a series of peaks plotted on a graph that measures signal intensity and mass
how do you analyze the results of a Tandem MS
starting at the right of the graph and moving left, each successive peak has one less amino acid than before. The difference in mass from peak to peak identifies which amino acid was lost in each case
