Please enable JavaScript.
Coggle requires JavaScript to display documents.
Protein practical methods - Coggle Diagram
Protein practical methods
chromatography
use buffered solutions, low temperatures and inhibitors to minimize denaturation and degradation
Solubility fractionation
use different pH values or elevated temperatures
very low slat concentrations are used for the differentiation of serum albumins and globulins
very high salt concentration (ammonium sulphate since it minimizes denaturation)
Techniques
Gel filtration
comparison of native protein size and quaternary structure size can be used to work out how many polypeptides are present per molecule
best with small amounts
protein mixture moves down the column
largest do not go into pores, and so go round which is faster
smaller proteins can go through pores, so move slower
The sample is placed on top of tightly packed column of beads and the sample flows down the column, driven by gravitational or hydrostatic forces or with aid of pump
Used to plot callibration curves using log(molecular weight)
separation based o: the proteins flow around the beads but they spend some time in the large depressions that cover the bead surface, and as smaller proteins can penetrate these depressions more readily than larger and so stay trapped for longer
Ion exchange
separation based on overall charge of molecule; if positively charged beads then negative charge (acidic proteins) adhere the rest move unimpeded
charged beads are used at bioloical pH
example- positively charged beads attract negative proteins; then increase salt concentration so more Cl- will stick to knock of the proteins
works well with large amount of proteins
The proteins are then eluded selectively from the column by passing salt gradient through column, the higher the charge on protein the higher the salt concentration required for elution
Uses modified beads with surfaces covered by amino (NH3+) or carboxyl groups (COO-) and so carry either positive or negative charge at pH7
Affinity chromatography
separates based on biological activity
example- carbohydrate biinding
protein separated by binding to a column with beads that have had glucose residues covalently attached to form an affinity matrix
proteins released when carbohydrate added
used on small scale due to complexity and ost
ligands or other molecules that bind to the protein of interest are covalently attached to the beads in column, so an affinity column will retain only those proteins that bind the molecules attached to the beads
The remaining proteins do not bind so a single protein can be isolated, however if a retained protein is in turn bound to other molecules then the entire complex will be retained
Bound proteins are then eluted by adding excess soluble form of ligand, by detergents, pH or salt concentrations
Absorbance
absorption of a photon excites electron to higher energy level
if too high or low it will not occur
needs to exactly match energy gap between 2 energy levels
energy change is associated with each transition determined by chemical composition and structure
molecular electronic transition can determine colour because of what is absorbed
spectrometer
white light goes through entrance slit, through a monochromator
the different colours go through the exit slit then through the sample to reach the detector
diffraction grating in the monochromatic determines the wavelength of light
can be quantified using the beer-lambert equation
A=E x c x l
so absorbance has no units
transmission falls exponentially with absorbance
Electrophoresis
it is used to separate dissolved proteins in an electric field based on molecular weight, charge and isoelectric points (charge to mass ratio)
gel retards larger molecule, so smaller molecules move further; and if have same mass adn shape the one with greater net charge moves faster
Types
SDS-polyacrylamide gel electrophoresis
it binds to hydrophobic residues at a near constant rate
residual structures are removed by reducing disluphide bonds
reduced SDS protein complexes act as linear molecules and covers charge so migration nearly only based on polypeptide size
Western Blotting is used to identify proteins after SDS
separated proteins transferred to membrane allowing antibodies to access them
primary antibody binds to target
secondary protein is a fusion of an enzyme to the antibody and bind to primary
reaction catalysed by enzyme produces a colour change (in process called chemiluminescence)
example
LUciferase is an enzyme present in fireflies and can be linked to an antibody
the presence of ATP and its substrate, luferin, luciferase catalyses a light emitting reaction
The intensity is proportional to the amount of enzyme-linked antibody and thus antigen in the sample
However, if the epitote is altered (e.g. partial folding) or blocked then antibody binding is reduced and presents inaccurate response
Antibodies generated by injecting short synthetic peptide based on protein of interest into animal or by attaching the epitope to an unrelated protein (epitope tagging)
The gell acts as a sieve allowing smaller species to manoeuvre more rapidly through its pores than larger species do
Gels are made by polymerising a solution of acrylamide monomers into polyacrylamide chain and simultaneously cross-linking chains into semisolid matrix; and so pore size can vary with polyacrylamide and crosslinking reagnent
if 2 or more polypeptides are crosslinked by disulphide binds then migration rate depends on whether protein has been reduced to break these bonds prior to electrophoresis- the crosslinked protein will appear larger
SDS is a detergent that denatures proteins (because it binds to hydrophobic side chains to destabilise hydrophobic interactions in protein core) and coats protein so is evenly negative charge (and move toward anode)
Immunoprecipitation
An antibody to the protein is added to sample and binds to epitopes on target protein
an agent binds to antibody is added to cause antibody and bound target to precipitate out of solution into particles isolated by centrifugation
Precipitate sobulised under denaturing conditions
If immunoprecipitated target is tightly bound to one or more other molecuels those bound molecules may be precipitated along with protein of interest
Two-dimensional gel electrophoresis
Proteins are separated by their charges and then their masses
SDS PAGE cannot readily resolve proteins with similar masses and so use their electric charge which is determined by pH of sample and pKa of ionisable groups on proteins as 2 unrelated proteins with simiilar masses are unlikely to have identical net charges so number of acidic and basic residues is different
Isoelectric focusing
a cell us fully denatured by high urea concentrations and then layered on gel strip with urea to remove bound SDS
The gel has a pH gradient (pH3-pH10) established using ampholytes
A charged protein placed at one end of the gell will migrate through the gradient until it reaches isoelectric point
To achieve the second dimension the gel strip is applied to SDS-PAGE to permit separation of proteins on basis of molecular weights
can be used to estimate molecular weight by comparing with standards of known weight
For linear polypeptides migration distance is proportional to log(molecular weight)
Isoelectric focusing
a gel with a pH gradient is used
proteins migrate to their isoelectric point
this has high resolving capacity and can be combined with SDS-PAGE in 2D electrophoresis
can then be placed on top of SDS slab to separate based on molecular weight
Mass spectrometry
MALDI-TOF
used to measure molecular weight directly
absorbed protein layer and matrix form crystals
a lazer is fired at them which desorbs and ionizes the proteins which are then analysed (accurate to 1Da)
Adding trypsin to protein (this hydrolyses peptide bonds at lys adn arg residues) can be used to theorise proteomes when genome is known
sample mixed with matrix (low molecular weight organic acid that absorbs UV)
TOF use the time it takes for an ion to pass through mass analyser before reaching detector, since it is proportional to sqrt of m/z
used to identify individual 2D spots from excised gel fragments (proteomic analysis)
Nuclear magnetic resonance spectrometry
can affect nuclei
distance between spots is used to find atom distance
used to observe flexible regions of proteins (X-ray cannot do this)
only for small proteins (80kDa) but does not require protein crystallisation
a concentrated protein solution is placed in a magnetic field and effects of differnet radio frequencies on nuclear spin states of diffeent atoms are measured
it can provide information about ability of a protein to adopt a set of closely related conformations and move between them
Key features
Ion source in whihc a charge, usually protons, is transferred to peptide or protein molecules under study causing them to be ionised
conversion of polypeptides to ions occurs in high electric field which directs charged molecular ions into mass analyser
mass analyser is always in vacuum and separates ions based on mass-to-charge ratios, separated ions directed to hit a detector which provided meaure of the ratio of each ion
There is also a data system to calibrate, store and process
Accuracy
detects 0.001-0.010 attomoles of a peptride or 1 attomole of a proteins of 200,000MW
error rates+/- 5ppm for peptides adn 0.05-0.1% for proteins
abundances of ions given are relative and not absolute values so to compare absolute values it is necessary to have an internal standard in sample; or by comparing 2 proteins mixed together in one MS, the mixing is possible due to m/z ratios in each sample being distinguished by MS from corresponding and chemically identical proteins in other sample (e.g. SILAC)
Crystallography
used to determine almost all known proteins
X-ray crystallography
as x-ray goes through sample it diffracts by electrons in the crystal
electron density map can be made
wavelengths are about 0.2-0.2 nm short enough to determine position of individual atoms
the precision with which one can determine the structure of a molecule from x-ray depends on quality of crystal used for analysis and flexibility of molecule in lattive
Cryoelectron microscopy
Used when proteins do not readily crystallise (like membrane associated proteins)
a dilute sample in an aqueous solution is applied in a thin layer to an electron microscope sample holder and readily frozen in liquid He to preserve structure
then examined using sensitive cameras using allow dose of electrons to prevent radiation induced damage to structure
computer algorithms analyse images to sort into same orientation
Centrifugation
2 particles with differnet masses or densities will settle to the bottom of a test tube at different rates
protein densities will not differ by more than 15% from 1.37g/cm3 unless attached to lipid or carbohydrate
Heavier or denser molecules settle more quickly than lighter or less dense molecules
purpose
preparative technique to separate materials with aim of obtaining enough to perform other experiments
an analytical technique to measure physical properties
sedimentation constant (s)
it is a measure of sedimentation rate commonly expressed in Svedberg units
Types
Differential centrifugaton
starting mixture like cell homogenate is poured in tube and spun at high speed
cell organelles like nuclei and large unbroken cells collect as pellet at bottom, soluble proteins remain in supernatant
supernatant and/or pellet can be subjected to further testing
Rate-Zonal centrifugation
a concentrated sucrose solution is used to form a density gradient, when a protein mixture is placed on top and subjected to centrifugation, each protein in mixture migrates down tube at rate controlled by its physical properties
samples are centrifuged just long enough to separate the molecules of interest into discrete bands
sedimentation rate can also be affected by shape, but rate-zonal centrifugation is a good practical method for separating differnet polymers
Radioisotopes
Half life is the time taken for half the atoms to undergo radioactive decay
Radiolabelled biosynthetic precursors are added to extracellular medium, they enter the cell via transporters and are incorporated into newly synthesised macromolecules
Methionine and cysteine labelled with sulphur-35, kinases can transfer phosphate-32 from phosphate-32 labelled ATP to label phosphoproteins
Labelled compounds have virtually same chemical properties as those they replace
Detection
The sample is overlaif with a photographic emulsion that is sensitive to radiation
development of the emulsion yields small silver grains whose distribution corresponds to the radioactive material
A Geiger counter measures ions produced in a gas by B particles and y rays mitted from radioisotope and is used to give quantitative measure
Phosphorimagers detect radioactivity using 2D array detector, storing digital data on the number of disintegrations per minute per pixel
Often used in combination with Gel electrophoresis in combination with autoradiography or phosphorimager analysis, the bands correspond to newly synthesised protein; individual proteins are isolated with immunoprecipitation, precipitation then solubilised and sample analysed
Pulse chase experiments
These are used ot trace chainges in intracellular location of proteins
a cell sample is exposed to radiolabelled compound for a brief period (pulse)
pulse ends when unincorporated radiolabelled molecules washed away and cells exposed to excess unlabelled compounds to dilute
samples periodically after the pulse are assyaed to determine location and chemical form