Protein Isolation and Purification Techniques

protein; isolation ;purification

Chromatography

Electrophoresis

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1.

. which can be obtained in large amounts and has..

2.

 high concentration of the protein

3.

. molecular cloning techniques allow production and purification

      of proteins from E. coli, yeast or other cells.

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1.

. serum proteins or secreted proteins are already soluble

2.

. otherwise cells must be broken up

•

osmotic lysis; perhaps aided by enzymes or chemicals

to   

         weaken cell membranes (detergents solvents, or

lysozyme for 

bacteria(

•

   mechanical disruption by grinding, blending, homogenizing

or

ultrasonic disruption

3

. filter or centrifuge crude lysate to remove cell debris (membranes, cell

walls etc

, 

)

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1.

. may be denatured by high temperature

•

keep solution at appropriate temperature, usually fairly cold  .

•

    can use denaturation of some proteins to help purify a protein which i

s

 stable 

at

high temperature

2.   Proteases are enzymes which break peptide bonds.

•

   maintain conditions which inhibit proteases ==> low temperature or change

in 

  pH or addition of chemical inhibitors

.

•

  may use proteases to digest labile proteins if the protein of interest is stable

to 

  proteases

.

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•

enzymes can be measured by the reactions they catalyze--either measure

products produced or reactants used u

p

•

.  other proteins may be measured by their biological effects: ability to bind

specific molecules or the effect of a hormone on cells, tissue or organism.

•

. Immunochemical techniques-can produce antibodies which bind specifically to

particular proteins

E. General Strategy of Protein Purification-

fractionate based on different characteristic

Proteins are purified by fractionation procedures in a series of steps.

1.

Charge

•

ion exchange chromatography

•

Electrophoresis

•

Isoelectric focusing

•

2.  Polarity

•

adsorption chromatography

•

paper chromatography

•

reverse-phase chromatography

•

hydrophobic interaction chromatography

3. Size

•

gel electrophoresis

•

gel filtration chromatography

•

ultracentrifugation

•

dialysis and ultrafiltration

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A.

Effects of Salt Concentration

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salting out

 –

at high 

concentrations of salt

,

solubility

 of the protein

decreased

 sharply (precipitate),because 

the salt ions bind most of the water

molecules

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.

B. Effects of pH

All proteins have an 

isoelectric point

, 

pI

, a pH at which they have no net charge, and

they are least soluble at their pI because there are no net electrostatic repulsions

between protein molecules:. Different proteins have different 

pI

's, so one can

manipulate the relative solubilities of a mixture of proteins by changing the pH.

C. Crystallization

Once a protein is reasonably pure, one may try to crystallize it which is the ultimate

criterion of purity.

•

   The number and distribution of charges,

•

 nonionic polar groups,

•

and hydrophobic residues on the surface of the protein

•

The size and shape of the protein .

•

determines the concentration of the salt needed to cause precipitation of the

protein.

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.

III. Chromatographic Separations

One of the most power class of separation procedures  is chromatography; this

technique can take various forms based upon the physical apparatus

column chromatography,

 paper chromatography,

 or thin layer chromatography –

. All depend upon having a 

mobile phase

, usually a liquid, and 

a stationary phase

,

usually a solid coated with a liquid. The sample is applied in the mobile phase which

passes down the column (or paper or thin layer plate) 

and the different solutes move at

different speeds depending upon their relative affinities for the mobile and stationary

phases; 

we say that they 

partition

 between the mobile and stationary phases.

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The Chromatographic Matrix

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.

(4) have large or small pores accessible to the protein

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          the conditions during sterilization.

•

A 

variety of materials have been used as matrices. These 

include

--inorganic materials 

glass

, 

silica

, and 

hydroxyapatite;

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1.

. Stationary Phase

 -- chemically bound charged groups with counter ions bound; these

may be positively charged groups which bind anio

n

)

anion exchanger

) or negatively

charged groups which bind cations )

cation exchanger

)

2

. Mobile Phase

 -- an aqueous buffer solution characterized by: pH and ionic strength

3

 . Eluting Ion Exchange Columns

 -- molecules usually adsorb tightly in the buffer in

which they're applied ==> must weaken this interaction.

•

Increase ionic strength (most common method): 

F = q

1

 q

2

 / D r

2

q

1

and q

2

 are the charges on 

2

groups, r is the distance between the groups, and D is

the dielectric constant of the solvent which is increased with higher ionic strength

thus weakening the force between the solute and the ion exchanger. Another way to

look at this is that other ions in the buffer compete for the ion exchanger binding

site.

•

change pH -- changes the charges on the molecules being separated; also can change the

charge of a weak ion exchanger

•

These changes can be made stepwise by changing the buffer reservoir (step gradient) or as

gradient -- by mixing two buffers

B

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1

. Column Packing

 -- spherical porous beads of defined size

crosslinked dextrans

 --

Sephadex (Pharmacia(

crosslinked polyacrylamide

 -- Bio-Gel P (Bio-Rad(

crosslinked agarose

 -- Sepharose (Pharmacia) or Biogel A (Bio-Rad). Agarose beads

have very large pores and are, therefore, good for separating very large molecules

other materials developed by other companies

2

. Gel beads 

are designed to have a distribution of pore sizes around a mean

pore size. The mean pore size and the distribution determines the size range of

molecules which can be separated.

3

. Dialysis 

is a form of molecular Filtration

.

C

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based upon specific binding of the target protein to a particular ligand which is

bound to an inert matrix.

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1

. Reverse Phase Chromatography

stationary phase is more hydrophobic liquid

adsorbed to inert matrix

mobile phase is more hydrophilic liquid

2

. Hydrophobic Interaction Chromatography

--similar to reverse phase

chromtography but with less densely packed hdrophobic groups ==> less

denaturing.

3

. HPLC = High Performance Chromatography

--a form of column chromatogrpahy

with very small particles in the stationary phase to increase resolution; speed

increased by using very high pressures. Commonly used in reverse phase mode

but also with ion exchange, gel filtration, or hydrophobic interaction.

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spleen cells cytoplasm was   applied on top of sepharose

6B gel filtration column. Flow rate was 30 ml /hrs, 5ml

fraction collected.

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•

Like Zonal sedimentation, need some way of stabilizing the zones to prevent

mechanical mixing (from vibrations) or convection mixing (from temperature

differences -- a particularly severe problem with resistance heating caused by the

electric field

.

Gel electrophoresis

 is a method used in clinical chemistry to separate proteins

by charge and or size (IEF agarose, essentially size independent) and in

biochemistry

 and 

molecular biology

 to separate a mixed population of 

DNA

 and

RNA

 fragments by length, to estimate the size of 

DNA

 and 

RNA

 fragments or to

separate 

proteins

 by charge.

 Nucleic acid molecules are separated by applying an 

electric field

 to move the

negatively charged molecules through an 

agarose

 matrix. Shorter molecules move

faster and migrate farther than longer ones because shorter molecules migrate

more easily through the pores of the gel. This phenomenon is called sieving.

Proteins are separated by charge in agarose because the pores of the gel are too

large to sieve proteins. Gel electrophoresis can also be used for separation of

nanoparticles

•

4- Gel  electrophoresis  media ;

•

- Three types

•

Starch Gel 

-- swollen potato starch granules (little used now except for prep

isoelectric focusing)

•

Agarose Gel 

-- purified large MW polysaccharide (from agar) ==> very open

(large pore) gel used frequently for large DNA molecules

•

Polyacrylamide Gels 

-- most commonly used gel because they are very stable

and can be made at a wide variety of concentrations or even with a gradient of

concentrations ==> large variety of pore sizes

•

Acrylamide Concentrations 

-- typically 5-20% by weight (5%, 7.5%, 10%, 12.5%,

15%, 20% are commonly used values) ==> gel is mostly water.

•

 Acrylamide polymerizes in head-to-tail fashion to form long polymers which

form a complex network held together by bis-acrylamide crosslinks. The cris-

crossing polymers create pores in the gel; the size of pores is determined by

the acrylamide concentraion.

•

5-

. Acrylamide can be polymerized into any desired shape -- two shapes used

for electrophoresis

•

Tube Gels 

-- polymerize in glass tubing ==> cylindrical shape

•

Slab Gels 

-- polymerize between glass plates _--

B. SDS PolyAcrylamide Gel Electrophoresis -- SDS PAGE

1.

Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate

: CH

3

(

))

(

CH

2

)

11

SO

-

3

Na

+

This is a detergent because it contains a hydrophobic region, the 

CH

3

)

CH

2

)

11

 tail,

attached to a hydrophilic group, SO

3

-Na+, making it 

amphipathic

. It is a very strong

detergent which 

denatures

 proteins by binding to the polypeptide 

backbo

ne

estimation of purity and molecular weight makes use of the detergent 

sodium dodecyl

sulfate (SDS).

                              Na+ - SO4(CH2)11CH3

SDS

 binds 

to most proteins in amounts 

proportional 

to the molecular  weight of the

protein, about 

one

 molecule of SDS for every 

two

 amino acid residues.

 SDS, net 

negative

 charge, rendering the intrinsic charge of the protein 

insignificant 

and

conferring

 on each protein a similar charge-to-mass  ratio.

Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on

the basis of mass (molecular weight).

Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) is the

most direct method for assessing in a fast and reproducible manner, the relative

molecular weight (Mr) of denatured polypeptide chains and the purity of a

protein preparation.

 In SDS-PAGE, the sample to be applied to the gel is first treated with the

anionic detergent SDS which denatures the proteins in the sample and binds

tightly to the protein molecules. The SDS molecules confer a relatively uniform

negative charge to the polypeptide in proportion to its length. When an electric

current is applied across the gel, all proteins will migrate through the gel matrix

toward the anode.

 In this way, SDS-PAGE separates proteins according to size because the SDS-

coated proteins have a uniform charge:mass ratio. Proteins with less mass travel

more quickly through the gel than those with larger mass because of the sieving

effect of the gel matrix

.

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1

. pI - IsoElectric Point

: pH at which a protein has a net 0 charge 

(positive and negative charges

balance). Depends mostly on the amino acid composition and a little on the tertiary structure

2

. Create a pH gradient in a gel:

 Can be done on a 

slab

 (vertical or horizontal) or a 

tube

.

the final positions of each band depend only on an intrinsic property of the proteins, their

pI

's, and not on where they started in the gel

D

. Two-Dimensional Electrophoresis:

There are many variations; all basically combine

 2

types of electrophoresis

Proteins Electrophoresis carried out in gels made up of the cross-linked

polymer (polyacrylamide).

The polyacrylamide gel 

acts as a molecular sieve, slowing the migration of

proteins approximately in proportion to their charge-to-mass ratio.

µ = V / E = Z / f

µ, The electrophoretic mobility of the molecule.

V, 

the velocity of the particle molecule,

E, 

moving force of the molecule(electrical potential)

Z, 

the net charge of the molecule.

f, 

frictional coefficient

.-----

 reflects in part a protein’s shape.

 Different PAA Concentrations for different purposes

NonSDS-PAGE , Denatured SDS-PAGE, Nondenatured

The proteins are visualized by adding a dye such as 

Coomassie blue

, which binds to

proteins but not to the gel  itself.

In compares' with the positions to which proteins of known molecular weight migrate

in the gel, the position of an unidentified protein can provide a measure of its

molecular weight.

If the protein has two or more different subunits, the subunits will generally be

separated by the SDS treatment and a separate band will appear for each

Reagents and Buffers.

Acrylamide-bisacrylamide(30%).

          dissolving 29.2 gm of acrylamide and 0.8 gm of bisacrylamide in 50 ml of

distilled water. When the acrylamide was completely dissolved, the volume is

completed to 100 ml with distilled water. The solution is stored in a refrigerator at 4°C

in a dark bottle for no longer than one month.

2-Concentrated Resolving Gel Buffer (1.5 M Tris-HCl, pH 8.8).

             dissolving 18.2 gm of Tris-HCl in 80 ml of distilled water. The pH  adjust to 8.8

with 1N HCl, and the volume  completed to 100 ml with distilled water.  It was stored

at 4°C in a refrigerator.

Most modern commercial equipment is

 color-coded 

so that the red

or positive terminal of the 

power supply 

is connected to the red lead

of the gel apparatus. The 

red lead 

goes to the lower buffer chamber.

The black lead 

is connected to the black or negative terminal and

goes to the upper buffer chamber.

In slab gel electrophoreses negatively charged proteins or nucleic

acids move to the positive electrode in the lower buffer chamber (an

anionic system).

Proteins separated in cationic (+) systems. In these gels,

the proteins are positively charged because of the very low

pH of the gel buffers (e.g., acetic acid/urea gels for histone

separations) or a cationic detergent (e.g., CTAB).

Proteins move toward the negative electrode (cathode)

the polarity is reversed compared to SDS-PAGE: the red

lead from the lower buffer chamber is attached to the

black outlet of the power supply, and the black lead from

the upper buffer chamber is attached to the red outlet of

the power supply.

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gel is connected directly to the outlets of a power supply, then these

gels are connected in parallel = voltage is the same across each gel.

power supply reads 100 V, then each gel has 100 V across its electrodes.

The total current, however, is the sum of the individual currents

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Two identical gels require double the current to achieve the same

starting voltages and electrophoresis separation times.

3-  Concentrated Stacking Gel Buffer (0.5 M Tris-HCl, pH 6.8).

  dissolving 6 gm Tris-HCl in 50 ml of distilled water. The pH was

adjusted to 6.8 with 1N HCl; volume was completed to 100 ml with

distilled water. Solution was stored at 4°C in a refrigerator.

4-  Sodium Dodecyl Sulfate Solution 10% (SDS).

  Prepared by dissolving 10 gm SDS in 50 ml of distilled water and the

volume was completed to 100 ml with distilled water.

5-  Stock Sample Buffer (0.06 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, and

0.025% bromophenol blue)

 mixing  of the following;

0.5 M Tris acid pH 6.8 …………………………..1.2 ml

10% SDS ……………………………………………..2.0 ml

Glycerol …………………………….........................1.0 ml

0.5% bromophenol blue (w/v water)......0.5 ml

Sucrose ……………………………………………….0.2 gm/ml

Water …………………………………………………4.8 ml

 The mixture was stored at room temperature.

6

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         prepared immediately before use by adding 0.5-1.0 µl/ml of the final solution volume.

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11:  Fixing Solution

      Prepared by dissolving 100 gm of tricholoracetic acid in 500 ml of distilled water, 400 ml of

absolute methanol was added, the volume brought to 1 L with distilled water.

12- Staining Solution.

    mix methanol, glacial acetic acid, and coomassie brilliant blue G-250 in a final

concentration of 45% with 9% and 0.05% respectivly.

13-Destaining Solution.

    mix methanol, acetic acid, and distilled water in final volumes of 1:4:5   respectivly.

1- Resolving Gel Preparation 10%

   mix the following volumes of the previously prepared stock solutions:  Acrylamide-

bisacrylamide (30%)  ……..5.0 ml

SDS (10%)………………………………………....0.1 ml

Resolving gel buffer …………………………3.75 ml

D. W. ...……….…………………………………….6.25 ml

 This mixture must be degassed under vacuum. Ammonium persulphate  and TEMED

must be added at the ratios of 50µl / ml and 5µl / ml of the final volume, respectively.

2- Stacking Gel Preparation

 prepare at concentration of 5% by mixing the following:

acrylamide-bisacrylamide (30%)….……...1.3 ml

SDS (10%) …………………………………………..0.1 ml

Stacking gel buffer …………...……………….…2.5 ml

D. W………… ………………………………………….6.1 ml

     The mixture shoud be degassed under vacuum. 50 µl of 10% of ammonium

persulphate and 10 µl of TEMED were added to each 10 ml of degassed monomer

solution immediately before casting.

3- Resolving Gel Casting.

The 10 % resolving gel was applied to the electrophoresis unit. Its surface was

immediately overlaid with water saturated butanol and lefted for 45 minutes to 1

hrs at room temperature to polymerize. butanol was removed after gel

polymerization,  the gel surface was rinsed with D.W.  The stacking gel was added

at the same way and lifted to polymeriz. The cast comb was then inserted in

between the glasses and removed after hardening of the stacking gel.

Phosphorylase 94 kDa

Albumin 67 kDa

Ovalbumin 43 kDa

Carbonic anhydrase 30 k Da

Trypsin inhibitor 20 kDa



- Lactalbumin 14.4 kDa

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