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

Used to plot callibration curves using log(molecular weight)

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

works well with large amount of proteins

example- positively charged beads attract negative proteins; then increase salt concentration so more Cl- will stick to knock of the 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

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

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

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)

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)

Mass spectrometry

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

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)

used to identify individual 2D spots from excised gel fragments (proteomic analysis)

Nuclear magnetic resonance spectrometry

can then be placed on top of SDS slab to separate based on molecular weight

Adding trypsin to protein (this hydrolyses peptide bonds at lys adn arg residues) can be used to theorise proteomes when genome is known

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

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

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

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)

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

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

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

Uses modified beads with surfaces covered by amino (NH3+) or carboxyl groups (COO-) and so carry either positive or negative charge at pH7

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

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)

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

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

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

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

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)

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

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