protein analysis

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  • Created by: Sarah
  • Created on: 02-06-18 12:57
how can you homogenisation of tissue?
pestle and mortar, blending, sonication
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how do you separate unbroken and broken material?
centrifugation
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why do we need to purify proteins
20,000-30,000 proteins in a cell
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how can you confirm protein identity when you know the protein?
western blot
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how can you confirm the protein identiy when you don't know the protein?
mass spectrometry
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how can you check the protein is pure?
gel electrophoresis
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what is the principle of differential centrifugation?
spin at different speeds getting higher and higher. 1st pellet has cells + nuclei. 2nd has mitochondria, lysosomes and perixosomes spin at very high speed pure cytosol + ribosomes
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what fraction do you take in differential centrigugation?
whichever one will have your protein of interest in
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what are the 2 types of density based ultracentrifugation?
velocity sedimentation + equilibrium sedimentation
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what does velocity sedimentation centrifugation separate by?
slow and fast sedimenting component in sucrose gradient
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how does velocity sedimentation work? difference between slow and fast sedimenting components?
form sucrose gradient, load sample spin at high speed. Fast sedimenting = heavier organelles. Slow sedimenting = lighter organelles (vesicles). Puncture hole and collect fractions
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what does velocity sedimentation sort by?
fast and slow sedimenting components
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what does equilibrium sedimentation support by?
low and high buoyant density
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how does equilibrium sedimentation work?
run sucrose gradient for long time (2hrs) and spin for long time (20hrs)
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what do you do after centrifugation?
chromatography
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what is chromatography?
separation of mixture by passing it through a medium where they will move at different rates
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what are the 3 types of chromatography?
1) gel filtration (size exclusion chromatography) 2) affinity chromatography 3) ion exchange chromatography
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what comes out of the intitial fractions in gel filtration? why?
larger proteins- they move faster through the column as they don't enter the beads and get retarded
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how does gel filtration work?
beads have pores. Beads packed in a column. Apply sample to column. A plug keeps the beads in place. Apply extra solvent to push it through by gravity then collect fractions
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what runs faster in gel filtration?
larger proteins
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what is the working range of the resin defined as?
the pore size of the beads as they have a sieving effect
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what does affinity chromatography rely on?
tight interactions of the protein with its binding partner
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how do you purify an enzyme?
affinity chromatography- attach substrate to beads (eg glucose) will capture enzyme
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what does the first fraction of affinity chromatography contain?
all of sample that doesn't interact with beads - eluted by applying more and more solvent
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how do you elute the captured material?
by adding more substate- add more glucose with compete with glucose on the beads
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how can a protein be captured by affinity chromatography?
can use an antibody on the bead, capture by immunobinding and then break interaction betwen antibody and POI
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how do you brerak the interaction between POI and antibody?
use very strong acid or high salt (breaks electrostatic interactions)
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what is the flow through in affinity chromatography?
everything that does not bind to the beads
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how is the bound protien eluted?
using a competing ligand to dislodge the affinity interaction
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what is an example of affinity chromatography that uses DNA/oligonucleotide?
attach DNA/oligonucleotide to bead putin column add extract elute unbound by solvent then apply high salt to elute DNA binding protein
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what can you do with affinity chromatography?
1) enzyme+substrate 2) antibody+ protein 3) DNA/oligonucleotide + DNA binding protein
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what does gel filtration seprate proteins based on?
size- large and small
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what does affinity chromatography separate proteins based on?
strength of interactions
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what does ion exchange chromatography purify proteins according to?
their charge
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why do we use positively charged beads usually in ion exchange chromatography?
proteins are usually slightly negatively charged
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what are put on the beads for ion exchange chromatography?
carboxy-methyl (CM) = cation exchange and DEAE (diethlaminoethyl) = anion exchange
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what is used for negatively charged beads?
CM
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what is used for positively charged beads?
DEAE
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how does ion exchange chromatography work?
load protein onto column and before applying. Some protein did not interact with the column(flow through) when baseline level increase conc of salt depending on strength of interaction will elute proteins. weaker interaction first
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what do you do after you elute specific proteins fraction by fraction in ion exchange chromatography? for enzymes
can track enzyme activity by doing reactions for each fraction and identify this fraction has desired enzyme activity- know enzyme is in that fraction
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what do you do after you elute specific proteins fraction by fraction in ion exchange chromatography? for protein
use antibodies & western blot to know the protein is in the fraction
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what does salt break?
electrostatic interactions
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what does the radioactivity binding experiment show?
the affinity of the binding partner to the protein of interest
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how does radioactivity binding experiment work?
radioactively label the binding partner and have your POI stuck to beads. Add different concentrations of the binding partner and measure radioactivity. the lower the concentration needed for the binding partner-higher the affinity. gives PO saturati
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what is increased to elute proteins from the column in ion exchange chromatography?
increasing salt concentration
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how is protien purified?
use all 3 chromatographys
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how do you show you've fully purified the protein?
SDS gel/gel electrophoresis
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what is SDS used for?
denature proteins- makes all proteins negatively charged thin sticks
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how do you do SDS gel electrophoresis?
load protein smaple on top of polyacrimide gel. SDS makes -vely charged so they will run in the current towards the anode
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what does SDS gel electrophoresis separate proteins by?
charge
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when SDS puts protiens into large negatively charged sticks it will be proportional to what
charge proportional to molecular mass
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what is the principle of SDS page?
SDS is -vely charged and hydrophobic. Proteins hide hydrophobic core but add hydrophobic molecule with charge protein will unfold them and reveal hydrophobic middle. So gives -vely charged sticks
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what has more SDS large or small proteins?
large proteins
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what moves faster in SDS page field?
larger ,molecules slower and smaller faster
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why is B-mercaptoethanol used?
to break disulfide bonds- separate each polypeptide chain separately
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what do you have to do to proteins in SDS gel?
boil then for 2 min in a solution containing B-mercaptoethanol and SDS
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what does 2D gel electrophoresis use?
isoelectric focusing in the 1st dimension
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what is 2D gel electrophoresis useful for?
complex samples
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what is isoelectric focusing in the first dimension?
run proteins in a pH gradient. Proteins seprate according to their charge because at a certain pH proteins lose their charge
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why doesn't 2D gel electrophoresis use SDS?
proteins have their own charge- gels allow you to seprate proteins by native charge
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what is the isoelectric point?
the point in the pH gradient where the protein loses its charge so no longer migrates in the electric field
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what do you do after isoelectric focusing?
run sample in isoelectric focusing in column, take gel and put it on top of the SDS denaturing sample and run it in another dimension = 2D gel pattern
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how do you separate unbroken and broken material?

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centrifugation

Card 3

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why do we need to purify proteins

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Card 4

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how can you confirm protein identity when you know the protein?

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Card 5

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how can you confirm the protein identiy when you don't know the protein?

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