Download - Pemurnian Protein
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TEKNIK ISOLASI PROTEIN Protein ekstraseluler
Tahapan isolasi Sentrifugasi cairan sel/ medium kultur menghasilkan :
Supernatan (crude extract) Pelet (sel & komponen non-protein)
Protein intraselulerTahapan isolasi :
Cuci jaringan/ sel dengan bufer salin : untuk menghilangkan pengotor/ bahan ekstraseluler Sel mikroba : sentrifugasi & resuspensi dalam bufer
Lisis sel memecah/membuka sel ekstrak : homogenat Menggunakan mortar/pestle, homogenizer, sonicator,
tissue grinder, cell disruptor, blender Menghasilkan : homogenat
Sentrifugasi menghasilkan : pelet (mengandung protein membran) supernatan (crude extract)
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Cell disruption methodsCell lysis method Kind of tissue
Blade homogenization Most animal, plant tissue
Hand homogenization Soft animal tissue
Sonication Cell suspention
French pressure cell Bacteria, yeast, plant cell
Grinding with alumina/ sand Bacteria, plant cell
Glass bead vortexing Cell suspention
Enzyme digestion Bacteria, yeast
Detergent lysis Tissue culture cell
Organic solven lysis Bacteria, yeast, plant cell
Osmotic shock Erythrocytes, bacteria
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Removal of Non-Protein Components
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PROTEIN SEPARATION
The goal of a protein separation is to obtain the protein in a
pure, active form using a minimum number of steps The shortest time possible.
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SEPARATION METHODChromatographic Methods
Proteins differ in size and chargeIon exchange chromatography
Anion and CationGel Filtration (size exclusion)
Separates based on sizeAffinity Methods
Affinity for ligandsEngineered affinity sites, e.g. histidine tag
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Chromatography
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Solubility techniquesSalting out methods
Solubility sensitive to ionic strength. Initial “salting in” and then“salting out” At high salt, solvent tied up with interacting with salts so that it is insufficient to solubilize proteins.
1st go to maximal salt that protein target is soluble, centrifuge and discard pellet. Now add just enough salt to bring down protein. Collect pellet.
Organic solvents Same principle as salting out; taking advantage of different
solubilities. Avoid totally denaturing proteins.pH
Proteins have many ionizable groups with range of pKs. When net charge of protein is zero, this is the isoelectric point or pI. Proteins are typically least soluble at their pI due to minimizing charge charge interactions.
Crystallization The solubility methods where proteins are ppt can be used to
grow crystals of proteins. This is only done when the protein is relatively pure
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Ammonium sulfate precipitation (salting out)
When high concentration of salt are present, proteins tend to aggregate and precipitate out of solution : “salting out”
Different proteins precipitate at different salt concentration. pH, temperature and protein purity play important roles in determining the salting out poin.
Ammonium sulfate is the salt choice because it combines many useful feature : salting out effectiveness pH versality high solubility low heat of solution low price
Ammonium sulfate concentration : % saturation Simple equation for calculation of gram ammonium sulfate needed to
make an X% solution from Xo% : 515 (X-Xo) g = ------------- 100-0,27X
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Dialysis
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ION EXCHANGE CHROMATOGRAPHYLow salt
High salt
P+ + Na+Na+ + P+
IONIC IONIC ELUTIONELUTION pH pH ELUTIONELUTION
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Ion exchange groups used in protein purification
STRONG CATION ─SO3
- ─ Sulpho
─ CH2SO3- ─ Sulphomethyl
─ C3H6SO3- ─ Sulphopropyl
WEAK CATION ─ COO- ─ Carboxy
─ CH2COO- ─ Carboxymethyl
STRONG ANION ─ CH2N+(CH3)3 ─ Triethylaminomethyl
─ C2H4N+(C2H5)3 ─ Triethylaminoethyl
─ C2H4N+(C2H5)2CH2CH(OH)CH3 ─ Diethyl-2-hydroxy- propylaminoethyl
WEAK ANION ─ C2H4N+H3 ─ Aminoethyl
─ C2H4N+H(C2H5)2 ─ Diethylaminoethyl
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Gel Filtration
Porous beads made of different materials. Size of pores can be controlled
Small molecules small enough to go into beads whereas larger go around
and thus flow faster. There is exclusion limit (all proteins too
large to go into pores).Can be used as preparative method or
be used to determine molecular sizeGels made of dextrans, agarose or
polyacrylamide
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Affinity MethodA ligand which has tight
binding to protein is attached to matrix.Protein of interest binds but
others pass throughElute using soluble ligand.Beside small molecules, can
also use antibodiesRecent advances in molecular
biology entails engineering a poly his group which binds to Ni or use parts of other proteins and columns that bind to this other protein
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Addition of glucose (G)
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Visualization &/or Isolation of a protein proteins migrate in an electrical field at rates
that depend upon their net charge, size, and shape Gel Electrophoresis... separation by charge (
separation of in a media as gels) in a media as porous gel (starch/polyacrylamide) : gels & staining
Isoelectric Focusing*: proteins are separated in a gel of a continuous pH gradient, proteins move to point in gel equal to its pI, i.e., no charge
SDS-PAGE*: Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis... proteins treated with ionic detergent that separates according to size SDS binds to protein @ 1 SDS/2 aa's thus proportional to a protein's MW
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SDS-PAGE
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Identification of protein’s presence & & its quantification
Identification - is often done by spectrophotometry spectrophotometers measures intensity of light beam before & after light passes through a liquid solvent with sample dissolved in it, (in a cuvette)... compares the two light intensities over a range of wavelengths.
Percent transmittance... ratio of intensity of light passing through the sample to the intensity of light shining on sample multiplied by 100%. Absorbance... is the log of the transmittance instruments... Spectronic 20/ spectrophotometer UV/ Vis
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SPECTROPHOTOMETRIC METHODS of DETECTING PROTEINS
UV absorbance at 280 nm. (measures aromatic aa's) Colorimetry reactions - colored dye binds to amino acids Ninhydrin reaction - rx's w amino = blue color (10-9
M) Biuret test = mg quantities… based on Copper ion binds stiochiometrically = violet color Bradford test = ug amounts based on dye Coomassie blue - binds to peptide Fluoroescamine dye = pg quantities... (10-12 M)
Quantification of amounts of protein present Quantification is based on BEER-LAMBERT Law linear relationship between... light Absorbance vs. Conc Protein Standard Curve
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Quantification of Protein Concentrations with ENZYME ACTIVITYrelating protein amounts & enzyme activity
1 (international) UNIT of ENZYME ACTIVITY… that amount of protein which converts 1 micromole of
substrate to product per min at 250C at optimal pH UREASE - 1 unit (IU) will liberate 1.0 µmole of ammonia from urea per minute at pH 7.0 at 25°C [equivalent to 1.0 I.U.] 1 UNIT of SPECIFIC ACTIVITY… the number micromoles converted per min per mg protein i.e., Units (as above) of enzyme activity per mg protein 1 UNIT of MOLECULAR ACTIVITY… number of units of enzyme activity per µmole
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Protein Purification