Salting out

Dialysis

Gel
Filtration

Protein Purification
Source - Bacteria, virus, plant or a mammalian Cell or
tissue

Homogenization

Separation
Properties used:
Charge
Hydrophobicity
Affinity
Solubility & stability
Molecular weight

Techniques:
Precipitation-salting
out
Centrifugation,
Dialysis
Chromatography
Electrophoresis

Chromatography- Gel exclusion or gel filtration, Ion

exchange, Affinity chromatography, Hydrophobic
interaction chromatography, High performance liquid
chromatography, etc.

Characterization
Gel electrophoresis
2-Dimensional gels
Immunoblotting
MALDI-TOF
amino acid analysis
protein sequencing
Mass Spectrometry, etc.

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

Salting
out
Fractionation of serum proteins using different

concentrations of Ammonium Sulphate (NH 4)2SO4.

Solubility of proteins is a function of the ionic

strength and pH of the solution.

Addition of salt at higher concentration to the
protein solution increases salt-solvent interaction
and decrease solvent-protein interaction.
Separates proteins from other impurities and also
fractionates them among themselves.
Different proteins precipitates at different salt
saturation.
Hydrophobic patches on protein surface plays
important role in precipitation

Initial concentration of Ammonium sulphate per cent

saturation

Final concentration of Ammonium sulphate Percent saturation

0
10
15
20
25
30
33
35
40
45
50
55
60
65
70
75
80
85
90
95

10

15

20

25

30

33

35

40

45

50

55

60

65

70

75

80

85

90

95

100

56

84

114

144

176

196

209

243

277

313

351

390

430

472

516

561

610

662

713

767

28

57

86

118

137

150

183

216

252

288

326

365

406

449

494

540

592

640

694

28

57

88

107

120

153

185

220

256

294

333

373

415

459

506

556

605

657

29

59

78

91

123

155

189

225

262

300

340

382

424

471

520

569

619

30

49

51

93

125

158

193

230

267

307

348

390

436

485

533

583

19

30

62

94

127

162

198

235

273

314

356

401

449

496

546

12

43

74

107

142

177

214

252

292

333

378

426

472

522

31

63

94

129

164

200

238

278

319

364

411

457

506

31

63

97

132

168

205

245

285

328

375

420

469

32

65

99

134

171

210

250

293

339

383

431

33

66

101

137

176

214

256

302

345

392

33

67

103

141

179

220

264

307

353

34

69

105

143

183

227

269

314

34

70

107

147

190

232

275

35

72

111

153

194

237

36

74

115

155

198

38

77

117

157

39

77

118

38

77
39

Why Ammonium
Sulphate?
Cheap
Easily soluble at higher amount
Easily available
Doesnt denature protein
Higher charge

Fractionation of proteins using different

concentrations of ammonium sulphate
[(NH4)2SO4]

Contd.

Dialysis
Separation technique that facilitates the removal
of small, unwanted compounds from
macromolecules in solution
Selective and passive diffusion through a semipermeable membrane
smaller molecules move from their higher
concentration to lower concentration till
equilibrium is achieved
Dialysis membrane is regenerated cellulose
Dialysis can also be used to change the buffer of
a protein
Reverse Dialysis can be used to concentrate the
sample

Dialysis

Dialysis

Gel filtration (Size-exclusion/Molecular Sieve)

chromatography
Separation is on the basis of molecular size and shape
protein sample is applied to the top of a column consisting of
porous beads
The pores size in the matrix determines the rate at which proteins
diffuse into the beads
Some proteins are completely excluded
Sephadex, Sepharose, and Bio-gel are commonly used commercial
preparations of beads
Small molecules can enter these beads, but large ones cannot
small molecules are distributed in the aqueous solution both inside
the beads
Large molecules are located only in the solution between the
beads
Molecules are eluted according to decreasing molecular weight
Longer the column, better the resolution

Gel filtration (Size-exclusion/Molecular Sieve)

chromatography

Gel filtration (Size-exclusion/Molecular Sieve)

chromatography
Preparation of slurry:

Contd.

Contd.