why would you need to purify protein? - siumed.edubbartholomew/-lectures/protein methods 08.pdfi....
TRANSCRIPT
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Why would you need to purify protein?
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Methods for Working with Protein
1. Protein IsolationA. Selection of a protein source
i. tissue and cell cultures (bacteria, yeast, mammalian, etc.)ii. genetically engineered - tagged proteins, over-expression
B. Solubilizationi. osmotic lysis - hypotonic solution
swelling and burstingii. French press - high pressure & small orificeiii. sonicatoriv. homogenizer -tissue grinder, v. glass beads versus mortar & pestlevi. dounce - .
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What properties of proteins can be used to separate and purify
them from each other?
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Methods for Working with Protein
2. Separation methodsA. Properties that are used to separate proteins:
i. chargeii. hydrophobicityiii. affinityiv. solubility & stabilityv. molecular weight
B. differential centrifugation - S-100 versus S-30C. precipitation/solubility
i. salting in versus salting outsolubility of a protein close to its pI versus the effect of salt interacting w/ solvent & not the protein
ii. examples of a. ammonium sulfate precipitation b. PEI - poly(ethyleneimine)precipitation
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S – solubility of protein in salt solutionS` - solubility of protein in pure water
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Protein Solubility
• Salt-in– At lower ionic strength, increased salt
increase solubility• Salt-Out
– At higher ionic strengths– Increased salt concentrations causes the
protein to precipitate out of solution– Competition between the added salt with
other dissolved solutes for solvation
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Solute-solute interactions > solute-solvent interactions
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Solubility of lactoglobulin depends on pH
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Methods for Working with Protein
2. Separation methodsD. chromatography - overall example
i. ion exchange - cation vs. aniona. strong versus weak (effect of pH) b. counterion present is importantc. types of gradients and their application
linear, nonlinear, step
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Methods for Working with Protein
2. Separation methodsD. chromatography - overall example
ii. affinity chromatographya. ligand based:
glutathione covalent linked to resinGST fusion protein
b. speciality dyes - Cibracon blue and othersc. Immunoaffinity:
epitope tags such as FLAG, V5, etc.d. DNA - general and specific DNAse. others (example: heparin, hydroxyapatite)
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Methods for Working with Protein
2. Separation methodsD. chromatography
iii. gel exclusion or gel filtrationa. separation based on sizeb. exclusion volumec. different size limit materials
iv. HIC or hydrophobic interaction chromatographycompare to reverse phase chromatography
v. types of resin cellulose, dextran, agarose, polyacrylamide, perfusion
beads
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Sequence Databases
Some specifically for proteins and others for DNA
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http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed&itool=toolbar
http://blast.ncbi.nlm.nih.gov/Blast.cgi
PubMed – access to multiple databases
BLAST search
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Gel filtration or size exclusion chromatography
What is the size of the protein of interest and the contaminating protein?
How best to separate them?
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Precautions or concerns might you have about maintaining your
protein’s activity
What can you do to avoid these problems?
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Methods for Working with Protein
3. Stabilization of proteinA. Changing buffer
i. dialysis ii. ultracentrifugation
B. Concentrating proteinhelps to maintain active proteinsometimes addition of a carrier protein may helpaddition of glycerol
C. Inhibitorsi. proteasesii. phosphatase inhibitors
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Methods for Working with Protein
4. Protein AnalysisA. Gel electrophoresis
i. Discontinous gel a. polyacrylamide:
ratio bisacrylamide/ acrylamideb. TEMED - free radical stablizerc. ammonium persulfated. stacking gel pH 6.8, contains glycine pK2=9.78e. running gel is pH 8.8, sample contains
bromophenol blue
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Methods for Working with Protein
4. Protein AnalysisA. Gel electrophoresis
ii. SDS-PAGEa. SDS forms a micelle around the polypeptide
the size and charge of the micelle is approximately proportional to the size of the polypeptide
b. denatures proteinsc. protein stains: Coomassie blue, silver stains,
fluorescent stains d. gradient gels
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SDS-PAGE –principle•Glycine is an amino acid, and its charge property changes depend on the pH.•In stacking gel (pH6.8), only a little amount of glycine is negatively charged, thus, glycinemoves very slowly. •SDS-bound proteins move much faster in low-density gel. Thus, proteins are stacked on the running front of glycine.•In separation gel (pH8.8), glycine is charged and moves very fast. Proteins can move dependent on their size.•Stacking gel 5%, pH6.8•Separation gel 6-20%, pH8.8-+
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Methods for Working with Protein
4. Protein AnalysisB. 2-Dimensional gels
• 1st dimension: isoelectric focusing tube gel format
• ampholytes to create an immobilized pH gradient• 2nd dimension is usually SDS-PAGE
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General Formula of Ampholytes
•Each ampholyte has a different pK and isoelectric point.•Each ampholyte has a particular buffering capacity
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Methods for Working with Protein
4. Protein AnalysisC. ImmunoblottingD. Gel-shift or EMSA
(electrophoretic mobility gel shift assay) assays
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Western blotting