Separation techniques

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Molecules can be separated:

Chemically: by charge, by action with specific reagents

Physically: by solubility, by molecular weight, by density, structure (e.g. shape)

1. By physical properties

- SDS-polyacrylamid-gellectrophoresis (charge) - Agarose-gellectrophoresis (charge) - Gelfiltration (molecular mass)

- Centrifugation (centrifugal force)

Separation

2. By chemical properties- Ion exchange chromatography- Affinity chromatography- Solubility/desalting- DNA/protein chips or microarrays

3. By physical-chemical properties

Native polyacrylamid-gelelectrophoresisPaper-electrophoresis2D-gelelectrophoresis

Centrifugation

1. Differential centrifugation - Pellet/supernatant2. Density gradient centrifugation - Continuous gradient - Step gradient

Gel filtration or size exclusion chromatography

Approximately half of the volume of GFC columns is occupied by the resin. Small molecules, which enter all the pores of the resin elute after one column volume. For large molecules, which can not enter any of the pores, at least half of the column volume is necessary. These molecules leave the column after half column volume.

Intermediate sized molecules can enter some of the pores, but not all – they elute between the two other fractions. Ball shaped (isometric) molecules are well separated, elongated ones are difficult to separate. Why?

Gel filtration does not showgood separation, but can be used for desalting or for the separation of molecules of very different size.

agarose

Agarose gel electrophoresis

Agarose is a polysacharid extracted and purified from sea moss. For separation of large molecules, such as proteins or DNA.

DNA stain

Agarose gel electrophoresis

Pulse field gel electrophoresis

Continuously changing the direction of the electric field. The large charged particles will not be trapped in the network of polyacrylamide. Very large molecules and particles, even chromosomes can be separated by this technique.

The picture shows the separation of chromosomes from different strains of Plasmodium, the parasite causing disease malaria.

Polyacrylamide electrophoresis (PAGE)

Native-PAGE

- Molecule mass- Charge

SDS-PAGE

- Molecule mass

- No charge difference between molecules- No secondary structure (with reducing agent)

+ SDS (sodium dodecyl sulphate)+ reducing agent (e.g. mercaptoethanol)

For separaton of small molecules, such as proteins or small DNA fragments.

Paper electrophoresis

Electrophoresis of proteins can be carried out on wet paper. Though paper electrophoresis does not have a good resolution. However, it is a valuable tool, e.g. it can be used for the separation of native proteins. (in SDS gel electrophoresis the proteins are in denatured state).

On this figure we see electrophoretograms of healthy and sick people.

Isoelectric focusing

e.g.A

e.g.B

A

Ph gradient

9

8

7

6

5

4

-

+

B

separation on pH-gradient by the charge

Forming a pH gradient:

With free amfolins orimmobilens (immobilens (weak acidic or basic molecules)

Amfolins - forms a pH gradient in electronic field

ImmobilensImmobilens -covalently bound to the polyacrylamide matrix

2D-electrophoresis

First dimension:Isoelectric focusing

Second dimension:Separation by molecule mass

Proteins in a breast cancer Proteins in a healthy breast

At slightly basic pH (8-9) most proteins are negatively charged and bind to anionic exchangers. When the ion concentration is raised, first those molecules are eluted which have the lowest number of negative side chains. Highly negatively charged

proteins can be eluted only with

high ionic strength solutes.

The figure shows that fractions eluting at different salt concentrations contain very different proteins.

Ion exchange chromatography of proteins

Ion exchange chromatography

ANIONS with exchangeable counterions CATIONS with exchangeable counterions

starting adsorbtion start desorbtion end desorbtion regeneration

buffer counterions separable materials gradient ions

Affinity chromatography

Affinity chromatography is a powerful technique to purify one component out of highly complex mixtures. It is based on the interaction of two molecules (biotin-avidin). One is fixed on the column, while the other is selected out of the mixture.

A special form of affinity chromatography is metal chelate chromatography, where complexes of heavy metal ions are fixed on the column. These columns have high affinity to proteins or peptides with numerous histidine residues. Elution is carried out by imidazol compounds (His is imidazol-alanine).

Affinity Chromatography

immobilization

Sample (S) adsorption

impurities

Desorption of the sample

Affinity separations

Affinity chromatography frequently uses antibodies. Another separation technique, which is based on the use of antibodies is magnetic separation. Proteins, organelles, even intact living cells can be separated with colloidal, plastic-coated magnetic beads, carrying antibodies on their surface. The figure illustrates the principle of the separation:

mRNA can also separated with Dynabeads coated with oligo(dT) primers. Why?

DNA/Protein chips or microarrays

For genome wide analysis. DNA chips contains single stranded DNA spots on a glass surface, capable for hybridization with the complementary strand of a cDNA clone reverse transcribed from a single mRNA. One spot represents one gene. There can be 50 000 spots or more on a glass surface. The whole genome can be screened => Genomics

In protein chips there are antibodies bound to the glass surface. Each spot recognizes different protein molecules. The whole Proteome can be screened. =>Proteomics