Purification of cell components Sergiev P.V. 1755 Disintegration - - PowerPoint PPT Presentation

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MSU & SkolTech

Purification of cell components

Sergiev P.V.

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Disintegration of samples

Lysis of bacterial cells Lysozyme – breakdown of cell wall Freezing/thawing cycles Grinding with aluminum oxide Sonication Pressure drop

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Disintegration of samples

Lysis of yeast Bead beater Glass beads Grinding in liquid nitrogen Lyticase - cell wall breakdown

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Disintegration of samples

Lysis of mammalian cells or tissues Dounce homogenizer Dismembrator Trypsin/EDTA separate mammalian cells

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Centrifugation

Separation of cellular components according to their density Methods of centrifugation differ in rotation speed volume of the sample arrangement of vials centrifugation media

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Centrifugation

Table top centrifuge – the main working horse in the lab Rotation speed up to 14 000 rpm Up to 2 ml samples Fixed angle rotors Used for precipitations (Nucleic acids and proteins) and phase separation

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Centrifugation

Medium speed preparative centrifuge Rotation speed up to 20 000 rpm Up to 1 l samples Fixed angle and swinging bucket rotors Used for cell separation, debris separation after cell lysis

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Centrifugation

Ultracentrifuge Rotation speed up to 90 000 rpm (200 000 in some cases) 100 ml - 50 ml samples Fixed angle and swinging bucket rotors Used for separation of

• rganelles and macromolecular

complexes

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Centrifugation

Ultracentrifuge Fixed angle rotors: pelleting down macromolecules G= r w

2

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Centrifugation

Ultracentrifuge Swinging bucket rotors: Separation of macromolecular complexes by their sedimentation coefficient in a density gradient of sucrose or glycerol

u= rS w

2

S = D 18

20,w 2Dr

h

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Centrifugation

Ultracentrifuge Ribosome separation by a sucrose density centrifugation polysomes Top Bottom

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Chromatography

General principle

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Chromatography

Gel filtration Porous media (sephadex, sephacryl) particles bigger when pores are passing faster small particles are retraded

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Chromatography

Hydrophobic Salt concentration

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Chromatography

Ion exchange Salt concentration

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Chromatography

Affinity

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Chromatography

Affinity chromatography media Ni NTA His6 glutation GST IgG Z-domain of

• S. aureus protein А

Protein А(G) IgG Streptavidin Biotin Specific antibodies antigenes Immobilization methods BrCN NH2 Hydroxysuccinimide NH2 Epoxyde NH2,SH Hydrazide CHO

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Ultrafiltration

Passage through the pores

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Preparation of specific biopolymer type

Enzymatic degradation of the unwanted polymer: DNase treatment RNase treatment Protease tratment Phenol deproteinization Ethanol precipitation of nucleic acids Trifluoroacetic acid protein precipitation

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Gel electrophoresis

General principle

• +

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Electrophoresis in agarose gels

Application Buffer Agarose gel Power supply Typical gel Separation of DNA and RNA 50-20 000 bp non-denaturing Visualization by UV illumination via intercalating dye fluorescence Gel formation by cooling the melted agarose solution

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Electrophoresis in acrylamide gels

Application Separation of DNA and RNA 10 – 3000 nt Usually denaturing conditions (urea) Gel formation by radical polymerization, initiator (persulfate) and catalyst (TEMED) needed Visualization by UV, radioisotope or fluorescent labeling, methylen blue staining

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Capillary electrophoresis

Up to 96 samples at a time

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Southern blotting

General principle DNA fragments separation Transfer to a membrane Cross-linking to the membrane Hybridization with radioactive or fluorescent probe Northern blotting – similar method for RNA detection

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Protein gel electrophoresis

Denaturing (Laemmli) gel

pH 6.8 large pores , pH 8.8 small pores , Gly Gly- small conductivity high electric field quick movement of proteins Proteins are concentrated at the border

• +

high conductivity small electric field slow movement of proteins

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Protein gel electrophoresis

Denaturing (Laemmli) gel

SDS

• - -
• - - - - -
• -
• - -

Anionic detergent (SDS) denature proteins and make them negatively charged proportional to the molecular mass of protein

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Protein gel electrophoresis

Staining methods

Coomassie, 50 ng

silver nitrate ng 1

SYPRO Ruby ng 1 Other fluorescent dyes

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Immuno (western) blotting

General principle

• +

Protein separation Electrotransfer Blocking the rest of membrane by BSA primary antibody binidng Secondary antibody (conjugate) binding Development by a specific reagent

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Immuno (western) blotting

Methods for protein band visualization Alkaline phosphatase

NH O P O O- O- Cl Br NH OH Cl Br NH O Cl Br NH O Cl Br Br Cl O NH

BCIP AP

• 2H

(NBT) blue precipitate

NH NH NH2 O O N- N- NH2 O O N N NH2 O- O- O- O- NH2 O O O- O- NH2 O O

Horseradish peroxidase luminol

ОН

• О2

HRP hv

light

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Isoelectric focusing

Separation of proteins by isoelectric (neutrality) point

рН рН

Alkali solution Alkali solution Acidic solution Acidic solution Ampholines small molecules with a range of isoelectric points Proteins are locating according to their isoelectric points

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2D gel electrophoresis

Isoelectric focusing Separation by charge and mass Gel electrophoresis

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Differential 2D gel electrophoresis

sample 1 sample labeling by isoelectric focusing Laemmli electrophoresis 2 Су5 Су3 labeling by

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Protein identification

Matrix assisted laser desorption ionization (MALDI)

gel piece with protein sample peptides trypsinolysis mass spectrometry

laser impulse blows the matrix + + + + +

matrix

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Protein identification

Matrix assisted laser desorption ionization (MALDI)

+ + +

+

• detector

sample time of flight (TOF)

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Protein identification

Matrix assisted laser desorption ionization (MALDI)

8 7 8 .5 90 2.5 1 490 .7 1 54 6 .8 1 1 25 .7 1 84 9.7 1 9 29 .9 6 87 .4 10 96 .6 16 1 2 .7 20 0 5.7 2 14 7 .8 1 19 8 .7 27 35 .1 20 9 7 .0 17 94.7 9 7 6.4 2 2 0 1 .1 286 9.3

In te n s. [a .u .]

Peptide mass set is unique characteristic

• f protein

it allows protein identification