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Protein Purification

The document discusses several classical and molecular techniques for protein purification including ammonium sulfate fractionation, dialysis, ion exchange chromatography, gel filtration chromatography, and affinity chromatography. It provides examples of using these techniques to purify a protein and calculate purification fold and specific activity. The document also covers SDS PAGE and 2D PAGE gels.

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Attila Malinik
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59 views

Protein Purification

The document discusses several classical and molecular techniques for protein purification including ammonium sulfate fractionation, dialysis, ion exchange chromatography, gel filtration chromatography, and affinity chromatography. It provides examples of using these techniques to purify a protein and calculate purification fold and specific activity. The document also covers SDS PAGE and 2D PAGE gels.

Uploaded by

Attila Malinik
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 17

5/28/2012

Protein Purification

Chapter 3
Part 2

Protein Purification Techniques

Are Based on Chemical Properties of Proteins:

1. Solubility

2. Ionic charge

3. Size

4. Ability to bind ligands

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Solubility of Some Proteins

Classical Protein Purification Methods


The Oldest: Ammonium Sulfate Fractionation

Clear
Supernate

Clear Cell
Free Extract
Cloudy

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Ammonium Sulfate Fractionation Table

Ammonium Sulfate Fractionation

Example Results: 0 – 20% ppt, 20-40% ppt, 40 -60%


ppt, 60-80% ppt and 80-100% ppt….where is your
protein?

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These Fractions Need to be Assayed


For:
1. Total Protein can be done by
a. Absorbance at 280 nm
b. Colorimetric tests for protein
Lowry Assay, Bradford Assay

2. Your Specific Protein


a. specific enzyme assay, or
b. specific binding assay, or
c. unique spectral property.

Example Results – each Cut in 100 ml of Buffer


(NH4)2SO4 Cut Total Protein Your Enzyme
Crude Extract 1.73 g 3,895 μmole/sec/ml
0 – 20% 452 mg 0.1 μmole/sec/ml
20 – 40% 323 mg 3,560 μmole/sec/ml
40 – 60% 541 mg 12 μmole/sec/ml
60 – 80% 329 mg 0.1 μmole/sec/ml
80 – 100% 78 mg 0.01 μmole/sec/ml
Total Enzyme Activity Specific Activity
Crude: 3.9 x 103 μmole/sec 2.25 μmole/sec/mg protein
20-40 Cut 3.56 x 103 μmole/sec 11.0 μmole/sec/mg prot
(91% orig. activity) 4.9X fold purified

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Example Results – each Cut in 100 ml of Buffer


(NH4)2SO4 Cut Total Protein Your Enzyme
Cell Free Extract 1.73 g 3,895 μmole/sec/ml
0 – 20% 452 mg 0.1 μmole/sec/ml
20 – 40% 323 mg 3,560 μmole/sec/ml
40 – 60% 541 mg 12 μmole/sec/ml
60 – 80% 329 mg 0.1 μmole/sec/ml
80 – 100% 78 mg 0.01 μmole/sec/ml

How did you get rid of the high conc of (NH4)2SO4 ?

Dialysis – Getting Rid of (NH4)2SO4 or Change Buffers

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Column Chromatography Principle

Columns Connected to Fraction Collectors


Tube from column
attached here

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Ion Exchange
Chromatography

Fraction Collector

Ion Exchangers

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Ion Exchange Chromatography

Gel Filtration or Size Exclusion Chromatography

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Gel Filtration Media

Gel Filtration or Exclusion Chromatography

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Gel Filtration Gives an Idea of Molecular Weight

Affinity Chromatography

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Purification Table

Units = μM/sec or mM/min = rate…Enzymes convert S  P


Fold Purification = (Specific Activity at Step) / (Sp. Act. Crude Extract)
Final Purification is 1,500 fold pure.

Polyacrylamine Gel Electrophoresis

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Gel Showing
Steps in
Purification

Purification of RecA
from E. coli.

SDS PAGE Gels

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SDS PAGE Gels  Molecular Weight of Proteins

Isoelectric Focusing

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2-D Gels Start with Isoelectric Focusing

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2-D Gels End with SDS PAGE

A 2-D Gel from E. coli Cytoplasm

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Molecular Methods of Protein Purification


1. Isolate the gene…restriction enzymes, separation on
agarose gels, insert gene into a plasmid behind an active
promoter (turns on gene), and with a “tag” (such has
six-histidines) or Maltose Binding Protein or other tag.
The tag makes this a fusion protein:
Protein-his-his-his-his-his-his or Protein-MBP
2. Insert the plasmid into a bacterium (usually E. coli) and
turn-on the promoter to express the fusion-protein in large
quantities (the protein can be 10-30% cell volume!).
3. Lyse cells, fusion-protein binds affinity column which
after binding and washing provides the fusion protein
essentially pure.
4. Cleave off the his tag, dialyze  pure protein.

Molecular Methods of
Protein Purification

New England BioLabs, Inc

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Production and Purification of Tescalcin

Total lysate

Unbound
Wash 1

Wash 2
Wash 3
Elute 1
Elute 2
Time after induction (hours)
KDa 0 0.5 1.0 1.5 2.0 2.5 3.0

115
93
50
36 His6-Tsc
29 His6-Tsc

21

Expression Purification
cDNA cloned in pET15b expression vector Mechanical lysis
E. coli (BL21) transformation Ni-NTA metal affinity chomatography
Induction with IPTG Elution with imidazol

by Erasmo Perera, FIU student

Things to Know

1. Methods to Purify Proteins and How they Work.

2. Calculation of Specific Activity and evaluation of protein


purification.

3. Testing for protein purity.

4. How to do 2D PAGE gels.

5. Molecular Methods of Protein Purification

17

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