1. Strategies to Purify Protein
Jeremy Berg of the Johns Hopkins University
John Tymoczko of Carleton College, Lubert Stryer's Biochemistry
Lubert Stryer, Peter Gianakopoulos ,Mitchell Guss, Rong-Huay Juang and ……….
חיפשתי , מצאתי ,שיניתי וחיברתי אני יורי רזניק מקיף "ח" אשדוד . לא מאמין שכבר סיימתי. אוף......

2. 4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן,
שיטות להפרדת חלבונים
1.השפעת ריכוזי מלח ( אמוניום סולפט ) על חלבונים שונים
In /Salting Out Salting
2.הפרדה על פי גודל:
כרומטוגרפית סינון בג'ל
דיאליזה
אלקטרופורזה ב- S.D.S
3.הפרדה על פי מטען (אלקטרופורזה , מחליפי יונים , השקעה ב- pI )
4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן,
הורמון-קולטן)
5.הפרדה על פי קוטביות

3. 1 1 3 2 4 2 3 4 4 4 How to Separate These Objects 6 5 6 5 6 5 5 Shape
Size
Density 1 3 4 6 7 8 9 10 11 12 2 5
wood stone cotton wood wood cotton stone wood stone cotton stone cotton 1 2 3
Shape 4
Size 6
cotton
wood
stone 4 8 5 8 4 6 7 8 5
Density 5 7 9 10 11 12
Different rolling speed
Different sedimentation
Sieving different sizes
Juang RH (2004) BCbasics

4. Basic Principles of Protein Purification
Cell
Organelle
Homogenization
Macromolecule
Small molecule
Cell
Debris
Amino acid, Sugar,
Nucleotides, etc
Nucleic
acid
Protein
Carbohydrate
(Lipid)
Ammonium sulfate fractionation
Size
Charge
Polarity
Affinity
אלקטרופורזה ,
מחליפי יונים ,
השקעה ב-PI
chromatography,
Salting-out
Gel filtration,
SDS-PAGE,
Dialysis
Affinity
chromatography
Juang RH (2004) BCbasics

5. Strategies to Purify Protein
I. Solubility
Salting out
Proteins are less soluble at high salt concentrations
The salt concentration at which a protein precipitates differs
1.השפעת ריכוזי מלח ( אמוניום סולפט ) על חלבונים שונים
In /Salting Out Salting

6. Salting out Ammonium sulfate (half saturated) Ethanol (90%)
“salting out” adding salt eg ammonium sulfate
salting-out effect
as [salt]  less H2O is available for hydration of protein
proteins will aggregate

7. Strategies to Purify Protein
II. Size
Dialysis
Useful for removing small molecules
Not an efficient means to separate proteins
Gel filtration chromatography
Good strategy for separating proteins of different sizes
Larger molecules elute earlier
2.הפרדה על פי גודל:
כרומטוגרפית סינון בג'ל ,דיאליזה , אלקטרופורזה ב- S.D.S

8. Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.

9. molecular weight, native
molecular sieve chromatography (gel filtration, molecular sizing)
light scattering
hydrodynamic (equilibrium centrifugation)
mix
large molecules first

10. Gel Filtration Chromatography
Gel Filtration Chromatography. A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.

11. Polyacrylamide Gel Electrophoresis.

12. Polyacrylamide Gel Electrophoresis
Polyacrylamide Gel Electrophoresis. The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel. The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

13. molecular weight, denatured
SDS gel electrophoresis
molecular weight, denatured
gel filtration in denaturing solvent

14. + SDS-PAGE cont’d Dithiothreitol -reducing agent
-breaks disulphide bonds between subunits
-S-S-
DTT
-S-H
HS- +

15. - - + detergent binds strongly to proteins approx. 1 SDS/2 a.a.
SDS-PAGE cont’d
detergent
binds strongly to proteins
approx. 1 SDS/2 a.a.
Sodium dodecyl sulfate - =
SDS -
Protein +
Proteins (subunits) become linear
Gives all ptns an equal mass : charge ratio

16. SDS-PAGE cont’d

17. Strategies to Purify Protein
III. Charge
Ion exchange chromatography
Based on a proteins charge density
Elute bound proteins with an increasing concentration of a counter ion (e.g. NaCl)
3.הפרדה על פי מטען
(אלקטרופורזה , מחליפי יונים , השקעה ב- pI )

18. אלקטרופורזה - Kabat & Tiselius (1939) +
Albumin a b g + -
Globulins
Electrophoresis - a molecule with a net charge will move in an electric field
The velocity of a molecule is described by  = Ez/f
v = velocity
E = electric field strength
z = net charge of a molecule
f = frictional coefficient (depends on both mass and shape)

19. Electrophoretic analysis of serum
Sample application
Separation by charge - - + +
Anode
Cathode
Albumin a b g + -
Globulins

20. מחליפי יונים
Ion-Exchange Chromatography. This technique separates proteins mainly according to their net charge.

21. Ion Exchange - charge CATION EXCHANGE
Mixture of proteins is applied to the negatively charged
-ve and neutral proteins pass in the void volume, +ve proteins stick
Excess of Na+ ions is added
+ve proteins are eluted
Could have used change in pH to elute the proteins
CATION EXCHANGE

22. Solubility  fractional precipitation
Solubility depends on temperature, pH, ionic strength
Increasing temperature generally increases solubility
of limited practical relevance in biology
Solubility depends on the pH
minimum at the pI - the isoelectric point – the pH when the net charge on the molecule is zero

23. Solubility with pH Minimum when pH =pI
pI is the pH at which the molecules have zero net charge

24. Strategies to Purify Protein
IV. Binding activities
Affinity chromatography
Covalently attach “bait” to resin
Pass extract over the column
Wash to remove non-specific and weak interacting proteins
Elute
Usually by passing “bait” molecule through column
4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן, הורמון-קולטן)

25. Affinity Chromatography
Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G).
Could have used change in pH to elute the proteins

26. Glutathione Column Purpose: To purify GST or GST fused proteins GST =
Glutathione = 3 a.a. peptide (Glu-Cys-Gly) =
has a high affinity for

27. Glutathione Cont’d
Supernatant (Day 1)
Wash w/ Solution A
Sepharose
column
Flow through

28. Elution Step to collect GST
Glutathione Cont’d
Elution Step to collect GST
10 mM Glutathione
-add in 1mL fractions
Look!!!
MORE
beer!
Collect
in fractions
Could have used change in pH to elute the proteins

29. 5.הפרדה על פי קוטביות Polarity :adsorption chromatography
hydrophobic interaction chromatography

30. chromatography

31. chromatography

32. amino acid analysis, chromatography

33. Transform into a good scientist.