methods of biochemical analysis

types of homogenisation

• mechanical - blender, polytron

• liquid - dounce homogeniser, forced through narrow space

• freeze/thaw - ice crystal formation will disrupt cells

• manual grinding -mortar pestle, frozen in liquid nitrogen

sonication

• uses high frequency waves

• on liquid and smaller sample volumes

what has the largest effect on g in differential centrifugation

• changing the speed has greater effect than changing radius

competing solutes function and examples

• added to precipitate proteins in ion exchange chromatography

• ag ammonium sulfate, acetone or polyethylene glycol

• some attract water coat better than others

crude separation methods

• ammonium sulfate precipitation

• centrifugation

• gel filtration chromatography

competing solutes qualities

• very soluble in water

• relatively non denaturing

• easy to remove

• not too viscous or dense

• cheap

• pure

dialysis

• semi permeable membrane of bag

• conc of solution inside of bag then buffer on outide

• passive transport across membrane

who discovered chromatography

• mikhail tsvet

basic principles of column chromatography

• sample is added to stationary phase

• flow of mobile phase - organic solvent

• different substances appear after different elution volumes

elution volume

• the amount of mobile phase required to make a specific solute move through a column until it is eluted

size exclusion chromatography

• stationary phase beads

• small molecules enter the aqueous space between beads

• larger molecules run through faster bc cant enter beads

retention time of smaller molecules in size exclusion chromotography

• larger

• because the smaller molecules will enter the beads

what is the stationary phase usually in size exclusion chromatography

• polymers

• polysaccharides

ion exchange chromatography

• positively charged proteins bind to neg charged beads

• neg charged beads will have small retention time

how is bound protein eluted in ion exchange chromatography

• inc conc of NaCl

• increases competition of binding

affinity chromatography

• protein of interest eg enzyme will bind to substrate or substrate analogue and will leave the column

• protein of interest eluted by adding a competitor molecule or changing conditions eg buffer pH or salt conc

how many steps is affinity chromatography

• 1

• but strong association requires competitor to be added or changing of conditions

types of affinity chromatography

• immunoaffinity

• immobilised ligand

• lectin based affinity

• immobilised metal affinity

lectin based affinity chromatography

• lectin binds glycosylated proteins

• sugars added for elution

immobilised metal affinity chromatography

• metal ions - Ni2+ - bind to recombinant proteins

• nitrogen acts as donor

• proteins contain poly histidine tag

how do the recombinant proteins in immobilised metal affinity chromatography have good affinity for the metal

histidine

polyacrylamide gel electrophoresis characteristics

• highly porous to liquids

• use of detergent molecule sodium dodecyl sulphate

• apply electrical field

• smaller molecules move faster

what does sodium dodecyl sulphate separate based on

• molecular weight mass

sodium dodecyl sulphate qualities

• hydrophobic tail

• hydrophillic head

• forms micelles in aqueous solution

• forms neg charged complexes with polypeptide chains in denatured form

relationship betweem molecular weight of a protein and relative mobility in SDS page

• logarithmic

are disulfide bridges affected by sds

• no

• sds only disrupts non covalent interactions

what cleaves the disulfide bridge in sds page

• reducing agent

• dithiothreitol (DTT)

reducing sds page

• ensures complete denaturation and separation of polypeptide chains

• molecular weight determination based on linearised form of each protein or subunit

• can reveal presence of disulfide bonds with comparison to non reducing sds page

isoelectric point

• pH = pI charge is zero

• due to zwitterions

relationship between pH and charge

• protein charge depends on pH

• low pH values will be pos charged

• high pH will become depronated, neutralised and neg charged

when is isoelectric focusing used

• 2d gel electrophoresis

• sds polyacrylamide slab

mass spectrometry

• band spot removed from gel

• isolated protein cut using protease

• peptide separation via chromatography

• peak investigated by mass spec - ionisation and fragmented in gas phase

• observed set of masses compared with predicted

use of mass spectrometry

• identify proteins from their mass spectra after proteolysis

• cells treated with / without drugs

• cancer / non cancer cells

• cells at different stages of development

immunoaffinity chromatography

• protein purification - monoclonal against protein of interest

• adsorption - highly specific purification

• elution - tight binding often via pH change

western blot analysis

• separated by gel electrophoresis

• transferred to polymer sheet incubated with antibodies

• unbound antibody is washed off

cytoskeleton immunofluorescence

• actin filaments with red fluorescent protein

• microtubules with green fluorescent protein

• nuclei with blue fluorescent dye (DAPI) that binds DNA