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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 intraseluler Tahapan 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)
Cell disruption methods Cell 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
Removal of Non-Protein Components
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.
SEPARATION METHOD Chromatographic Methods Proteins differ in size and charge Ion exchange chromatography Anion and Cation
Gel Filtration (size exclusion) Separates based on size Affinity Methods Affinity for ligands Engineered affinity sites, e.g. histidine tag
Chromatography
Solubility techniques Salting 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
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
Dialys is
ION EXCHANGE CHROMATOGRAPHY Low salt P+
+ Na+
Na+ + P+ High salt
IONIC ELUTION pH ELUTION
Ion exchange groups used in protein purification
─SO3-
STRONG CATION ─ Sulpho
STRONG ANION ─ CH2N+(CH3)3 ─ Triethylaminomethyl
─ CH2SO3-
─ Sulphomethyl
─ C2H4N+(C2H5)3
─ C3H6SO3-
─ Sulphopropyl
─ C2H4N+(C2H5)2CH2CH(OH)CH3 ─ Diethyl2-hydroxypropylaminoethyl WEAK ANION ─ C2H4N+H3 ─ Aminoethyl
─ COO-
WEAK CATION ─ Carboxy
─ CH2COO-
─ Carboxymethyl
─ Triethylaminoethyl
─ C2H4N+H(C2H5)2 Diethylaminoethyl
─
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 size Gels made of dextrans, agarose or
Affinity Method A ligand which has tight
binding to protein is attached to matrix. Protein of interest binds but
others pass through Elute using soluble ligand. Beside small molecules, can also use antibodies Recent 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
Addition of glucose (G)
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
SDS-PAGE
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
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
Quantification of Protein Concentrations with ENZYME ACTIVITY relating 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 Purification