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1. Chapter 3 (part 2)
Protein purification and Analysis

2. Why purify proteins?
• Pure proteins are required to study
enzyme function
• Pure proteins are required for structural
analysis (x-ray crystallography, NMR
spectroscopy)
• Pure proteins are required to obtain amino
acid sequence

3. Steps in protein purification
• Develop assay
• Choose source of protein
• Prepare tissue extract
– cell disruption
– subcellular fractionation
• Protein fractionation (several steps)
• Determination of purity

4. Differential Centrifugation
1000 g
tissue
homogenate
transfer
supernatant
Pellet
unbroken cells
nuclei
chloroplast
transfer
supernatant
transfer
supernatant
10,000 g 100,000 g
Pellet
mitochondria
Pellet
microsomal
Fraction
(ER, golgi,
lysosomes,
peroxisomes)
Super.
Cytosol,
Soluble
enzymes

5. Chromatography

6. Gel Permeation
Chromatography

7. Ion-exchange
Chromatography
- - - - - -
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
- - -
++++++
++++++
++++++
low salt buffer high salt buffer
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
Cl-
+ +
+ +
+
+ +
+
+ +
- - - - - -
- - -
Cl-
Cl-
Cl- Cl- Cl-
- - -
+ +
+ +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
+ + + +
+
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
- - -
- - -
++++++
++++++ ++++++

8. Affinity Chromatography
Add excess ligand

9. SDS poly acrylamide electrophoresis (PAGE)
SDS = H3C-(CH2)10-CH2-OSO3
-
SDS – denatures protein
coats w/ negative charge
-
- - -
-
-
- - -
-
-
-
-
--
-
Used to determine protein MW
And purity of protein prep

10. Isoelectric Focusing
pH 9 -
-
Decreasing pH +
Decreasing pH
+
pH 3

11. 2-D Electrophoresis
Decreasing MW
large
small
Decreasing pH
-
+
SDS-PAGE
Decreasing pH
Decreasing MW

12. Amino Acid Analysis
H
N C S H3N C
O- N
O
C
R
S
C
HN
C
C
H
O
R
1) Acid hydrolyze protein
2) Treat with phenylisothiocyanate (PICT)
+
3) Separate derivatized AA’s by HPLC

13. Protein Sequencing
(Edman Degradation)
H
N C S H3N C
O
C
R
H
NH C
O
C
R
X
S
N C
H
HN C
O
C
R
H
NH C
O
C
R
X
+
Trifluoroacetic acid
N
S
C
HN
C
C
H
O
R
H
2HN C
O
C
R
X
1)
2)
3)
Repeat
Can sequence in 30 to 60 AA’s from N-terminus

14. Generate Proteolytic
Fragments
Endopeptidases
•Typsin cleaves at COOH end of Lys and Arg
•Chymotrypsin cleaves at COOH end of Phe, Tyr, Trp
Chemical Cleavages
•Cyanogen Bromide cleaves at COOH end of Met
Generate overlapping fragments
Sequence individual fragments and piece together sequence

15. Peptide mapping exercise
Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp
Trypsin
Met-Ala-Arg
Phe-Ala-Glu-Gln-Asp
Gly-Glu-Tyr-Met-Cys-Lys
Chymotrysin
Met-Ala-Arg- Gly-Glu-Tyr
Met-Cys-Lys –Phe
Ala-Glu-Gln-Asp
CNBr
Met
Ala-Arg-Gly-Glu-Tyr-Met
Cys-Lys-Phe-Ala-Glu-Gln-Asp

16. Proteomic Analysis

17. Matrix Assisted Laser Desorption Ionization Time of Flight
(MALDI-TOF)