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17 Cards in this Set
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3 Analytical identification by charge/mass
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1. Electrophoresis
2. Isoelectric focusing 3. 2D gel electrophoresis |
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Column chromatography purification
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By ion exchange, size exclusion, or affinity
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Electrophoresis
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Mobility = total charge/friction= distance
Friction = mass Direction of movement depends on the charge |
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SDS gel electrophoresis(PAGE)
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1. Remove the charge so only mass counts
2. denature protein 3. encapsulate in SDS micelles to give a uniform charge 4. gives protein size 5. Compare mobility to a standard to get estimated mass |
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Isoelectric focusing
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Using a pH gradient
1. Put ampholytes into one end of the gel 2. Electrophores them and let them migrate to their respective pHs 3. Put in your protein and electrophores them 4. They will migrate to their isoelectric point at their pH |
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2D gel electrophoresis
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Seperating the molecules first by charge and then by mass with SDS.
This allows us to find abnormal proteins in disease states |
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Proteomics
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trying to identify all proteins in an organism
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Metabolomics
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Trying to identify all metabolites in an organism
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Acute phase response
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General response of the body to injury or infection of tissue.
Production of some plasma proteins increase like; C reactive protein, protease inhibitors(antitrypsin), blood coagulation proteins, alpha 1 and 2 globulins Other proteins decrease: albumin, transretin |
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CRP(C reactive protein) as a marker?
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A marker because it is a major component of acute phase response to bacterial infection
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Affinity chromatography
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Having a specific ligand for the target protein then later disrupting that bond to purify it
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Size exclusion
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Larger ones pass through more quickly because the need to slip through the cracks
The smaller ones can take their time and also diffuse through the porous proteins making it easier and slower |
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UV absorbance
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Directly proportional to the concentration of Phe, Tyr and Trp at 260-280nm.
The T's are more absorbant than P |
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DNFB and Dansyl chloride
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Dinitroflourobenzene gives flourescent N terminals and detects picomolar amounts
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Ninhydrin reaction used as a detector?
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Detects nanomolar amounts
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Edman(endo) degradation
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Breaks down proteins in repetitive cycles using endonucleases. They are then seperated by chromatography
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Proteolytic enzymes and targets, chart of of the enzymes targeting AA bonds?
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1. Trypsin - Lys, Arg
2. Chymotrypsin - Tyr, Phe, 3.Ile, Leu, Val, Trp, His 4.Pepsin - Phe, Leu 5.Thrombin - Arg 6.Papain - Arg, Lys, Phe 7.Carboxypeptidase - C terminal residue 8. Use endoproteases to sequence proteins |