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Chapter 3 (Part 2) : Protein Purification and Analysis

To study protein function and structure, pure proteins are required and must be isolated from cells or tissues. The process of protein purification involves disrupting cells, separating subcellular components through differential centrifugation, and using chromatography techniques like gel permeation, ion exchange, and affinity chromatography. The purity of protein fractions is analyzed using SDS-PAGE, isoelectric focusing, 2D electrophoresis, amino acid analysis and N-terminal protein sequencing. Proteolytic digestion generates protein fragments that can be sequenced and pieced together to determine the full sequence. Modern proteomic techniques like mass spectrometry are also used to analyze complex protein mixtures.

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Dawlat Salama
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11 views

Chapter 3 (Part 2) : Protein Purification and Analysis

To study protein function and structure, pure proteins are required and must be isolated from cells or tissues. The process of protein purification involves disrupting cells, separating subcellular components through differential centrifugation, and using chromatography techniques like gel permeation, ion exchange, and affinity chromatography. The purity of protein fractions is analyzed using SDS-PAGE, isoelectric focusing, 2D electrophoresis, amino acid analysis and N-terminal protein sequencing. Proteolytic digestion generates protein fragments that can be sequenced and pieced together to determine the full sequence. Modern proteomic techniques like mass spectrometry are also used to analyze complex protein mixtures.

Uploaded by

Dawlat Salama
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Chapter 3 (part 2)

Protein purification and Analysis

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

Steps in protein purification


Develop assay
Choose source of protein
Prepare tissue extract
cell disruption
subcellular fractionation
Protein fractionation (several steps)
Determination of purity

Differential Centrifugation
transfer
supernatant

1000 g

tissue
homogenate

10,000 g

Pellet
unbroken cells
nuclei
chloroplast

transfer
supernatant

transfer
supernatant

100,000 g

Pellet
mitochondria

Pellet
microsomal
Fraction
(ER, golgi,
lysosomes,
peroxisomes)

Super.
Cytosol,
Soluble
enzymes

Chromatography

Gel Permeation
Chromatography

Ion-exchange
Chromatography
+++

---

+++

----+ + +++
+ ++ + +
++
+ ++
+ ++ + +
+ ++
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++

low salt buffer

high salt buffer

++
+- ++- -+ +
+ ++
---

+ -+
Cl
Cl+- + +- + Cl- +
++
+ +Cl
+
+ +
+
Cl+- + +Cl+
+
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++

++
+ ++ + +
+ ++

+++

+++

++
- +- +++ +
+ ++
++
+ ++ + +
+ ++
+++

---

---

---

Affinity Chromatography
Add excess ligand

SDS poly acrylamide electrophoresis (PAGE)


SDS = H3C-(CH2)10-CH2-OSO3SDS denatures protein
coats w/ negative charge
--- -- - - - -- ---

Used to determine protein MW


And purity of protein prep

Isoelectric Focusing

pH 3

Decreasing pH

Decreasing pH

pH 9

2-D Electrophoresis

Decreasing MW

large

Decreasing pH

Decreasing MW

SDS-PAGE

small

+
Decreasing pH

Amino Acid Analysis


1) Acid hydrolyze protein
2) Treat with phenylisothiocyanate (PICT)
N

H3N

S
C HN

O-

C
C
O

3) Separate derivatized AAs by HPLC

Protein Sequencing
(Edman Degradation)
N

H3N

NH

2)

C HN

NH

Trifluoroacetic acid

3)

C HN
N

C
C

2HN

Can sequence in 30 to 60 AAs from N-terminus

Repeat

1)

Generate Proteolytic
Fragments
Endopeptidases
Typsin
Chymotrypsin

cleaves at COOH end of Lys and Arg


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

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

Proteomic Analysis

Matrix Assisted Laser Desorption Ionization Time of Flight


(MALDI-TOF)

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