PURIFICATION AND METHODS

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22 févr. 2014 (il y a 3 années et 3 mois)

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PURIFICATION AND METHODS

FOR STUDYING

MACROMOLECULES OF LIFE

Aleksander

L.
Sieroń

Department

General and molecular biology

And genetics

http://biolmolgen.slam.katowice.pl

Homogenization

(disintegration of cells and tissues)

To release:


Proteins


Nucleic acids

Homogenization

(disintegration of cells and tissues)


Homogenization


mechanical procedure


Sonication (disruption of cells by high
frequency sound
).


Passing the cells through narrow
oppening

under high pressure.


Permeabilization



making „holes” in cell
membrane using gentle detergent.


Pressuring cells using tightly fitted rotating
pestle in glass cylinder.


Fragmentation of a tissue using metal balls
and high frequency shaking.

Homogenization by shaking with metal
balls

SONICATION

Teflon pestle with
tube
-
like glass
cylinders

ISOLATION OF NUCLEIC ACIDS

Initial material (biological):


Peripheral blood;


Epithelial cells


Cultured skin fibroblasts


Amniotic fluid cells (AFC),
kosmówki

(CVS)


Hair follicle cells


Blood spots, semen spots, bone marrow, tissue
fragments, skull bone fragments, bone fragments,
tooth fragments.

DNA

isolation methods

1.
DNA isolation by
phenol:chloroform

mixture
extraction (used for removal of proteins).

2.
DNA isolation with salting out proteins from
lysed cells.

3.
DNA isolation using a DNA binding resin to
which DNA will be reversibly bound.

RNA

isolation methods

1.
Isolation of specific RNA (by total cell RNA
fractionation).

2.
Direct isolation of specific RNA (the method is
restricted exclusively to the cells synthesizing
particular RNA at increased amount).

3.
Poly(A)RNA isolation

4.
Isolation of RNA from

polyribosomes.


5.
Total cell RNA isolation with subsequent isolation of
particular RNA (cDNA


complementary DNA) by
Polymerase Chain Reaction
-

PCR .


Laboratory micro centrifuge

Columns with resin

Quantitative and qualitative analysis

After the nucleic acids, DNA or RNA, the first

step is determination of their purity and
concentration. Both DNA and RNA concentration
can be determined either by UV light
absorbance or by comparison of
ethidium

bromide fluorescence of the sample to control
of known concentration

Agarose

gel


DNA after PCR

Lanes 1 and 9 contain DNA
mass ladder

Agarose

gel
-

RNA

Chromatogram
-

BIOANALYZER

RNA

investigation methods


Northern
-
blot method


RT
-
PCR


RT
-
qPCR


SAGE (serial analysis of gene expression)


Microarrays


Hybridisation

in situ (microscopic technique)


RNAseq
: High through put
transcriptome

sequencing

DNA

investigation methods

Restriction enzymes

Hybridization (
renatur
ation
):
Southern Blotting, DNA
fingerprinting, Northern blotting,
In situ hybridization
(FISH).

DNA amplification by PCR.
Visualization of PCR
products can be achieved by
electrophoresis: PCR
-
Multiplex; PCR
-
RFLP (analysis of restriction
fragments length polymorphism)

Sequencing by enzymatic Sanger’s method

Parallel sequencing
-

Next Generation Sequencing

Chromatogram
-

sequencing

Sequencing

Polyacrylamide gel

RESTRICTION

Eco
RI

meth
y
lase

MODYFICATION

RESTRICTION

Eco
RI

restriction endonuclease

No cut by
Eco
RI


restriction endonuclease

Agarose

gel


result of restriction reaction on DNA

Transfer („blot”) DNA fragments from
agarose

gel onto membrane

NORTHERN BLOT

1.
The isolation of biological material:

a)
fragmentation of the material,

b)
the crushing of tissues and cells (homogenization,

c)
ultrasound break),

d)
extraction of the aqueous solution of the protein and removal of
residual material by centrifugation or filtration

2.
Pre
-
treatment (optional): precipitation of proteins from
solution as a result of fractionation of increasing amounts of
solvent or salting out with ammonium sulfate and removing
the protein precipitating agent by means of dialysis,
centrifugation, differential centrifugation, sedimentation,
speed, sedimentation equilibrium

3.
Purification: Chromatography

4.
Changing the ion (optional): dialysis

5.
Analysis of purity of the preparation: electrophoresis,
spectrophotometry.

Isolation steps in proteins purification

Density gradient centrifugation

Force of
centrifugation

Velocity equilibrium

Stable
sucrose
gradient

Equilibrium sedimentation

Layers containing particular
components can be collected

Type of
Chromatography

Separates Proteins By

Bind With

Elute With

Affinity

A specific interaction

No competing ligand

Competing ligand
(specific); conditions
that disrupt
protein/protein
interactions (non
-
specific)

Ion Exchange

Net surface charge

Low ionic strength

High ionic strength;
Increased (
cation

exchange) or
decreased (anion
exchange) pH

Hydrophobic
Interaction

Hydrophobicity

High ionic strength

Low ionic strength

Size Exclusion

Hydrodynamic radius

The four most common types of column chromatography
used in protein purification

Examples of selective and non
-
selective forms of affinity chromatography with the
functional group (ligand) used for specificity and typical elution conditions.
Summarized from Thermo Fisher Pierce and GE. See below for the common suppliers
for different types of columns based on a
Labome

survey of over 200 publications.

Protein to
Purify

Ligand

Elute With

Antibody (antigen
-
specific)

Antigenic peptide

Free peptide

Polyhistidine
-
tagged

protein

Ni
2+

or Co
2+

Imidazole or free
histidine

FLAG
-
tagged protein

FLAG
-
specific antibody

FLAG peptide or low
pH

GST
-
tagged protein

Reduced glutathione

Free glutathione

Myc
-
tagged protein

Myc
-
specific antibody

Low pH

Antibody (class
-
specific)

Protein A , G, or L

Extremes in pH

DNA
-
binding protein

Heparin

High
ionic

strength

The net charge on a protein is influenced by the pH of its solvent. At pH=
pI
,
the protein has zero net charge and, therefore, will not bind to a
cation

exchange or an anion exchange stationary phase. Adjusting the pH above or
below the
pI

of the protein will lead to a net charge, and protein binding to
either an anion exchange (pH >
pI
) or a
cation

exchange (pH <
pI
) stationary
phase.

COLUMN LIQUID CHROMATOGRAPHY

Size

exclusion

column
chromatography used in protein
purification

Hydrophobic interaction
chromatography. At high ionic strength,
proteins are partially
desolvated
, causing
them to adopt alternate conformations in
which normally buried hydrophobic
residues are more exposed. These
residues can then form hydrophobic
interactions with the hydrophobic
functional groups conjugated to a matrix.
Lowering the ionic strength causes the
protein to refold into its native
conformation, burying its hydrophobic
residues. This decreases hydrophobic
interactions between the protein and
stationary phase, facilitating protein
elution

Protein eluted from a hydrophobic interaction column with a decreasing salt
(ammonium sulfate) gradient. Fractions were analyzed for both total protein
content and activity specific to the protein of interest. The peak centered at
fraction 45 contains the protein of interest, as indicated by protein activity.
From http://www.insectscience.org/9.04/

Polyacrylamide SDS gel electrophoresis


SDS
-
PAGE

Two
-
demensional

polyacrylamide gel electrophoresis

Polyacrylamide gel
electrophoresis
aparatus

SDS
-
PAGE of samples collecting during a protein purification scheme. Gel is stained
for the visualization of all proteins.

From
http://www.omicsonline.org/ .

Schematic of a gel for dis
-
continous

gel electrophoresis. Electrolyte composition and pH value
of both gels as well as their porosity are different.

Protein separation according to their isoelectric points

Concentrating gel

Separating gel

Buffer


e.g. running buffer

Buffer for loading samples
-

sample buffer

Dye

front

Identification of proteins


Sequencing following:



Fragmentation to shorter fragments (selective
protease)



Protein fragment analysis by mass spectrometry

Analiza

danych

przez

porównanie

z
bazami

danych

(Blast,
Swissprot
)

Sample preparation

Gel map

Dot recovery

Selective protein cutting

Mass spectrometry

Protein identification

Data analysis by comparison

with databases (Blast,

Swissprot
, etc.

Pattern analysis

e.g.
PDQuest

Peptide profile from mass spectrometer

Protein study methods


Crystal formation








Roentgen
crysta
l
ography

X
-
ray di
f
fraction

(analogy to optic micros
k
op
e
)

Eye

Model of a molecule

Crysta
l
lographer

X
-
ray diffraction

Synchrotronic

radiation

At wave length of X
-
ray range

(0.1 nanometer)

Crystal

Detector

Computer

Computer assisted three
-
dimension

Distribution of electrons:

Positions of atoms are
reflected by high electron
density

Phase

information

Protein study methods


NMR spectroscopy (Nuclear Magnetic
Resonance)

NMR schematic

Area and
PCr

line
depends on metabolite
concentration

Line structure is linked to
molecule conformation

Chemical shift of line Pi
depends on pH

Phosphate 31P NMR spectrum from forearm muscle obtained in vivo. Lines in the spectra correspond
to nuclei 31P in
phospho
-
ceratine

molecules (
PCr
), inorganic phosphate (Pi) and ATP molecule.
Difference of chemical shifts of lines
PCr

and Pi enables pH measurement.

Sample

Magnet

Generator




Impulse

program
a
tor




Detector





Computer

Backbone model with side
chains

Ribbon
-
like
model

Filed Model

Flowchart for the comparative modeling process.

Three Polish scientists were among the winners of the sixth edition of the
prestigous

world competition in the modeling of the spatial structure of proteins, which takes
place in the United States under the name Critical Assessment of Techniques for Protein
Structure Prediction (CASP).

Theoretical

Model

Solved

Structure

Flowchart of a typical

Protein Structure Initiative (PSI)

pipeline

CD methods

Circular
dichroism

(CD) Spectroscopy enables:

1.
Determining how the protein is folded (characteristics of
secondary, tertiary and even quaternary structure of the
protein).

2.
Comparing the structure of proteins obtained from
different sources.

3.
Comparing the conformation of the proteins in the various
solutions and/or their thermal stability.

4.
Stability study of the conformation of proteins under
stress (thermal stability, stability at different pH, in various
solvents).

5.
Search for protein conformation changes under the
influence of ligands attached.

Characterization of secondary, tertiary and
quaternary protein structures.

Comparison of protein structure derived from
different sources
.

thermalstability

Thank you

Aleksander

L.
Sieroń

Department

General and molecular biology

And genetics

http://biolmolgen.slam.katowice.pl