Davletov Protein Structure, Analysis + Interacions

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  • Created by: Sarah
  • Created on: 27-02-18 15:23
what are the 2 types of density based ultracentrifugation?
1) velocity sedimentation (slow/fast sediment) 2) equilibrium sedimentation (buoyant density)
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in edman degradation what does PITC react with and form?
PITC reacts with amino acid group at the N terminal. Forms PITC derivative of the terminal residue (aa)
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what are the pH changes in edmann degradation?
1) coupling PITC at alkaline 8.6 2) add strong acid to break the 1st peptide bond
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why is only the 1st peptide bond is broken in edman degradation?
PITC attached to th ealpha amino acid destabilises the 1st peptide bond only so when strong acid is added its only the 1st aa lost
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how is the terminal aa seprated from the rest of the polypeptide in edman degradation? why?
organic solvent because it will be hydrophobic versus hydrophillic polypetide
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the 1st step of the protein purification is tissue homogenization what is this?
a biological sample is brought to a state where all fractions of the sample are equal in composition. Cellular disruption, technique to lyse cells to release their contents.
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what can be used for tissue homogenisation?
sonication (using sound energy), blending, pestle and mortar
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what technique is used after tissue homogensiation to seprate released from the unbroken material?
centrifugation
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what are the 6 typical steps in protein purification?
1) tissue homogenisation 2) centrifugation 3) chromatography 4) electrophoresis 5) westen immunoblotting 6) mass spectrometry
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why is electrophoresis uses in protein purification?
to confirm the protein is pure
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what is used in protein purification to confirm the identity of a protein?
western immunoblotting and mass spectrometry
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what is mass spectrometry?
measures the mass of charged particles
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in edman degradation what is used to identify its fraction standard on the graph?
high performance liquid chromatography
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what is differential centrifugation used for?
seperate organelles from the whole cell. Spin pellets to get different fractions with different organelles
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what happens in velocity sedimentation?
spin at very high speed in a sucrose gradient (dense sucrose at the bottom). Separates at fast/slow sedimenting component. From fast sedimenting can get fractions. Collect drop by drop. At first get high desne sucrose with nothing in then organelles
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what does the fast sedimenting component contain?
heavier organelles (weighs it down)
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3 types of chromatography?
1) gel filtration 2) ion exchange 3) affinity
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what type of chromatography uses beads that act as a sieve and slow down smaller proteins?
gel filtration
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describe gel filtration chromatography
apply a sample (With POI) to a column packed with beads. It has a plug to keep beads in place. Applying extra solvent pushes material down the column. Larger proteins come out first, smaller proteins retarded come out in later fractions.
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what happens in equilibrium sedimentation (a type of ultracentrifugation)
run sucrose gradient for a long time spin for 20 hours. Separated based on its buoyant density. Can then do further fractionation
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what does affinity chromatography rely on?
tight interactions of a given protein and its partner
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how does affinity chromatography work?
attach substrate to beads in a column to capture the protein. Other material will just flow through the column so 1st non interacting material released. Then apply more solvent and free the captured material by applying a competitive ligand
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if you instead use an antibody against a protein in affinity chromatography how does this work?
stick antibody to bead to capture protein. (immunobinding). Then elute it by braking the antibody interaction by using strong acid or high salt solution
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why do you use high salt solution in affinity chromatography?
high salt solution
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whats an example of a use for affinity chromatography?
purifying a DNA binding protein
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how do you purify a DNA binding protein?
attach a specific DNA sequence or oligonucleotide to a bead. Pass nuclear extract (stuff in nucleus) through the column -> elute unbound using solvent. Then use high salt to break interacting DNA binding from the DNA sequence
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what disease do you get if hydroxylation goes wrong?
scurvy on collagen
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in acetylation acetyltransferases work on what residue?
lysines
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what is acetylation important in?
histones, epigentics, tubulin, stabilises microtubules
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methylation happens on what?
lysines and arginines
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whats methylation important for?
epigentics and closes up DNA to TFs histones
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what do you get in the later fraction of affinity chromatography?
DNA binding protein
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Card 2

Front

in edman degradation what does PITC react with and form?

Back

PITC reacts with amino acid group at the N terminal. Forms PITC derivative of the terminal residue (aa)

Card 3

Front

what are the pH changes in edmann degradation?

Back

Preview of the front of card 3

Card 4

Front

why is only the 1st peptide bond is broken in edman degradation?

Back

Preview of the front of card 4

Card 5

Front

how is the terminal aa seprated from the rest of the polypeptide in edman degradation? why?

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Preview of the front of card 5
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