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Analytical Techniques > Extraction science > Flashcards

Extraction science Flashcards

To introduce the experimental techniques and principles of extraction and separation science for preparation and characterisation of biological samples. Prof Shaw

1
Q

Extraction sciences and sample collection

A

PH, buffers, and cell homogenisation

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2
Q

Separation science

A

Centrifugation, dialysis, lyophilisation, precipitation

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3
Q

Chromatography

A

Gel filtration, ion exchange chromatography, affinity chromatography

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4
Q

Protein separation and purification

A

Electrophoresis

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5
Q

Proteomics and metabolomics analysis

A

Protein sequencing, genome, transcriptome and metabolome

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6
Q

Femtomoles

A

10-15

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7
Q

Attomoles

A

10-18

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8
Q

Cell homogenisation techniques

A

Non Mechanical and Mechanical

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9
Q

Cell homogenisation - non mechanical

A

Osmotic shock Freeze-thaw Lytic enzymes Lysozyme

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10
Q

Cell homogenisation - mechanical

A

Pestle + mortar, abrasives Ball mills and glass beads Blenders and rotor stators Homogenisers Solid Extrusion Liquid Extrusion Ultra-sonication (>20 kHz)

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11
Q

Osmotic shock

A

Animal soft tissues Some plant cells Small scale only

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12
Q

Freeze thaw

A

Animal soft tissues Some bacteria Time consuming; small scale; closed system- suitable for pathogens with appropriate safety measures; some enzymes are cold-liable

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13
Q

Lytic enzymes

A

Animal cells, Plant cells, Mild and selective; small scale; expensive; enzymes must be removed once lysis is complete

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14
Q

Lysozyme

A

Some bacteria Gram-negative bacteria must be pre-treated with EDTA. Suitable for some organisms resistant to mechanical disruption

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15
Q

Pestle and mortar + abrasives

A

Tough tissues Not suitable for delicate tissues

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16
Q

Ball mills + glass beads

A

Bacteria and fungi May cause organelle damage in eukaryotes

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17
Q

Blenders and rotor-stators

A

Plant and animal tissues Ineffective for microbes

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18
Q

Homogenizers (glass & Teflon

A

Soft delicate tissue e.g. white blood cells, liver Glass may shatter- wear safety glasses during use

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19
Q

Solid extrusion (Hughes press)

A

Tough plant material; bacteria; yeasts Small scale

20
Q

Liquid extrusion (French pressure cell)

A

Microbial cells Small scale

21
Q

Ultrasonication

A

Microbial cells Cooling required; small scale; may cause damage to organelles, especially in eukaryotic cells

22
Q

Cell disruption - type dependencies

A

Animal cells No cell wall. Plasma membrane and cytoskeleton weak. Can be easily disrupted by using Waring Blender Plant cells Cellulose cell wall and additional lignin or waxes Can be disrupted by blenders or Press-solid extrusion or enzymes eg pectinases and cellulase Bacterial Cells Gm+ and Gm- bacteria- cell wall. Peptidoglycan Can be disrupted by sonication or using the enzyme lysozyme Fungal Cells Filamentous Fungi and Yeasts Robust- 90% polysaccharide (Chitin, mannan + protein microfibrils) Can be disrupted by grinding with glass beads, acidic washed sand or celite

23
Q

Disruption media

A

Buffer to replace intracellular buffer system. Usually ~pH 7.0 Inorganic salts. KCl or NaCl. Usually below 100mM Sucrose. Used to prevent osmotic lysis of organelles eg. Mitochondria or lysosomes. Also used to stabilise proteins. Mg2+- integrity of membranes – counteracting negative charge of membrane phospholipids. Also ATP in complex with Mg2+ EDTA (ethylene diamine tetra acetic acid). To chelate heavy metal contaminants that would otherwise inactive cysteine amino acids which are often important for protein stability and activity. Removes Ca2+ which can activate some proteases and nucleases. Protease inhibitors.eg. PMSF (phenylmethylsulfonyl fluoride- serine protease inhibitor). Intracellular proteases are released from lysosomes on cell breakage (lysosome acid pH so at neutral pH less active). Reducing agents. eg 2-mercaptoethanol, dithiothreitol (Clelands reagent). Prevent oxidation of proteins especially with free cysteine thiol groups which are needed for activity. They may be oxidised to disulfide bridges or sulfenic and sulfinic acid which can inactivate the protein. Detergents cause dissociation of proteins and lipoproteins from the cell membrane. SDS (sodium dodecyl sulfate) will denature proteins and is used for SDS-PAGE. Some non-ionic detergents such as Triton X100 are used for membrane protein isolation since they do not denature protein.

24
Q

Proteins and their separation

A

An understanding of the structure and function of proteins is important to understand since they make up a large proportion of the living cell. They are important for 1) Enzymes to carry out all of the reactions going on in the cell; 2) Structural proteins make up connective tissue, muscle and bones; 3) The Immunoglobulins (IgG, IgM, IgA, IgE) are part of the humeral immune response; 4) Proteins form complexes with RNA in ribosomes and DNA in nucleosomes; 5) Proteins interact with lipids in the cell membrane and act as transport systems into the cell; 6) Proteins interact with sugars in glycoproteins and most proteins in higher cells are glycosylated.

25
Q

Properties of proteins - MCHDM

A

Mass - most sensitive and specific of techniques, mass spec

Charge – pH

Hydrophobic/Hydrophilic Properties

Differential Solubility

Mobility in Applied Fields

26
Q

Protein isolation

A

Study of protein structure and function requires them to have a preserved quaternary structure They cannot be denatured To isolate proteins from a cell and keep under conditions close to the at of the cell; pH, ionic strength, viscosity, solvation, temperature Protect against degradation by proteases Preservation of their oxidation state - over-oxidised Separation and purification based on their molecular properties

27
Q

Amino acid properties

A

Non polar + hydrophobic:

Alanine, isoleucine, leucine, methionine, phenyalanine, proline, tryptophan, valine

Charged + polar:

Arginine, histidine, lysine, aspartic acid, glutamic acid

Polar (hydrophilic) + uncharged

Asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine

Charged + polar

Arginine, histidine, lysine, aspartic acid, glutamic acid

28
Q

Measures of charge, pH, pKa and pl

A
  • pH is more complex than just a measure of the hydrogen ion concentration, especially in complex fluids
  • The formal definition of pH is:

pH=-Log(a_(H_3O^+) )

  • Log is the Base 10 logarithm
  • a_(H_3O^+)is the activity of the hydrated hydrogen ion
  • Activity = g[H_3O^+]
  • gis the activity coefficient takes values 0 – 1
  • Activity reflects the non-idealityof a liquid such as the cytoplasm
29
Q

Interpreting pH and pKa

A
  • Lower pKa the higher Ka(because of the minus sign) hence
  • STRONGER ACID
  • pKa of an acid is the pH at which it is exactly half dissociated
  • pH > pK_aacid is fully dissociated
  • pH < pK_aacid is predominantly HA
  • Strong Acid HCl,Ka= 107
  • Weak Acid MeCOOH,Ka= 1.74 × 10-5

•

30
Q

Acid base equilibria

A
31
Q

Hendersen Hasselbach equation

A
32
Q

Measurement of pH

A

pH sensitive electrodes

Glass combination electrode

Electrode incorporates a pH sensitive layer and a reference Ag/AgCl electrode

33
Q

pH measurement

A
34
Q

Poly-Acid Titration – Phosphoric Acid

A
35
Q

Isoelectric Point

A

Isoelectric Point (pI)

  • The Isoelectric Point is the pH at which the molecule contains no net electrical charge
  • The protein may still be charged attracting a solvation shell
  • The protein may still be polar
  • The net charge – equal numbers of positive

and negative charges

• Incorporation into lipid bilayers may be favoured/required

36
Q

Ion Selective Electrodes

A
37
Q

Controlling pH with Buffers

A
  • Used to control pH of a solution
  • Usually a mixture of weak acid and conjugate base
  • Defined as- one which resists a change in H+ concentration (pH) on addition of acid or alkali.
  • In whole cells the cellular constituents act as buffers. eg proteins which have a large number of weakly acidic and basic groups as amino acid side chains.
  • Extent of pH change is determined as buffer capacity. This can be measured experimentally by a titration curve. The mid-point of the plateau of the sigmoidal curve is the pKa where there is best buffering capacity
38
Q

Buffers – change in pH

A

Add 10 mL of NaOH to 1L of water. What is the pH change?

pH of water is 7, Kw = [OH-] [H3O+]

[OH-]= 0.01moles in 1.01 L = 0.0099 M

So [H3O+] = 10-14/(0.0099) = 1.01 × 10-12

= pH 11.99

A pH change of 5 by adding only 10 mL of NaOH to 1L

39
Q

Acetate Buffer

A
40
Q

Buffer titration curve

A
41
Q

Blood buffer - carbonic acid

A
42
Q

Properties of ideal buffer

A

Impermeable to biological membranes

Biological stability and lack of interference with metabolic and biological processes

Lack of significant absorption of ultraviolet or visible UV light

Lack of formation of insoluble complexes with cations

Minimal effect of ionic composition or salt concentration

Limited pH change in response to temperature

43
Q

Example Buffers

A
  • Citric and Phosphate form insoluble complexes with divalent cations. Phosphate can act as a substrate, activator or inhibitor of certain enzymes.
  • TRIS often toxic to biological systems and can penetrate membranes decoupling electron transport systems in whole cells or isolated organelles.
  • Markedly affected by temperature with a tenfold increase in H+ concentration from 4oC to 37oC.
  • Zwitterionic buffers eg HEPES
  • Interfere with protein determination eg. Lowry assay.
  • Overcome many of the problems associated with other buffers since structure based on amino acids so more ‘Biological’.

BUFFERS WILL WORK BEST AT PH VALUES ONE UNIT EITHER SIDE PKA

44
Q

Separation of proteins

A
  • Mass
  • Centrifugation
  • Passage through membranes - dialysis
  • Affinity
  • Hydrophobic/Hydrophilic Properties columns • Differential Solubility extraction buffers
  • Charge and Mobility in Applied Fields • Buffer control
  • Separation on gels
  • Electrophoresis

• Single Cell Analysis

45
Q
A

Analytical Techniques (6 decks)

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