Analytical Techniques In Biochemistry-Extraction Science

Front 1

What has been the biggest challange of producing the human plasma proteome?

Back 1

Retaining the structural integrity of proteins whilst extracting each and every one

Front 2

10^ liters of what is PicoLitres?

What is the volume of an average human cell?

What is the average ATP concentration of an average cell?

Back 2

10^-12L

2pL

10mM

Front 3

How many genes are present in the human genome?

Of these, how many are needed?

Back 3

25,000 genes in total, about 18,000 needed

Front 4

Roughly, how many proteinsare found in the human body?

Roughly, how many metabolites are present?

How many forms of sugar are present?

Back 4

1,000,000 proteins

3,000 metabolites

10,000 sugars

Front 5

Of the cell homogenisation techniques, state 3 non-mechanical ones?

Back 5

Answer can include any 3 of the following:

-Osmotic shock

-Freeze thaw

-Lytic enzymes

-Lysozymes

Front 6

Of the cell homogenisation techniques, state 5 mechanical ones?

Back 6

Answer can include any 5 of the following:

-pestle and mortar+abrasives

-Ball mills and glass beads

-Blenders and rotor stators

-Homogenisers

-Solid extrusion

-Liquid extrusion

-Ultra-sonication

Front 7

Osmotic Shock:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 7

Animal soft tissues, some plant cells

Small scale only

Front 8

Freeze/thaw:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 8

Animal soft tissues, some bacteria

Answer can include:

Time consuming, small scale, some enzymes cold-liable

Front 9

Lytic enzymes:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 9

Animal cells and plant cells

Answer can include:

Small scale, expensive, enzymes must be removed once lysis is complete

Front 10

Lysozyme:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 10

Some bacteria

Gram negative must be treated with EDTA

Front 11

Pestle, motar and abrasives:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 11

Tough tissues

Not suitable for delicate tissues

Front 12

Ball mills and glass beads:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 12

Bacteria and fungi

May cause organelle damage in eukaryotes

Front 13

Blenders and rotor stators:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 13

Plant and animal tissues

Ineffective for microbes

Front 14

Homogenisers (glass and Teflon):

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 14

Soft, delicate tissues

Glass may shatter-goggles needed

Front 15

Solid extrusion (Hughes Press):

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 15

Tough plant material, bacteria and yeast

Small scale

Front 16

Liquid Extrusion (French Pressure cell):

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 16

Microbial cells

Small scale

Front 17

Ultrasonication:

What type of cell (s) would it be suitable for?

State a disadvantage of using this techniques.

Back 17

Microbial cells

Answer may include:

cooling required, small scale, may cause damage to organelles, especially in eukaryotic cells

Front 18

There are 8 components needed in a disruption media. State 6 of these.

Back 18

>Buffer-replace internal buffer,usually pH 7

>Inorganic salts-KCl and NaCl, usually below >100mM

>Sucrose-prevents osmotic lysis of organelles

>Mg2+ ions-improve integrity of membranes

>EDTA

>Protease inhibitors

>Reducing agents

>Detergents

Front 19

State the purpose of having:

EDTA

Protease inhibitors

Reducing agents

Detergents

In a Disruption Media?

Back 19

>EDTA: chelate heavy metal contaminations, remove ionic Ca2+

>Protease inhibitors: Prevent breakdown of proteins (use PMSF)

>Reducing agents: Prevent oxidation of proteins by disulfide bridges, sulfenic and sulfinic acids

>Detergents: cause dissociation of proteins and lipoproteins from the cell membrane

Front 20

State the 5 properties of proteins that can be measured.

Back 20

Mass

Charge-pH

Hydrophobic/hydrophilic properties

Differential solubility

Mobility in applied fields

Front 21

To isolate a protein from a cell, conditions within the cell have to be replicated. What 5 things need to be controlled?

What also does it need protection from?

What also needs to be preserved?

Back 21

pH, ionic strength, viscosity, solvation and temperature

Proteases

Maintain oxidation state

Front 22

There are 8 non-polar hydrophobic amino acids. State 5 of them.

Back 22

Answer can include any 5 of the following:

Alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine

Front 23

There are 7 hydrophilic, un charged, polar, amino acids. State 5 of them.

Back 23

Answer can include any 5 of the following:

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

Front 24

There are 5 charged, polar amino acids. State 2 positively charged ones and 2 negatively charged ones.

Back 24

Positively charged:

(Answer can include)Arginine, histidine and lysine

Negatively charged:

Aspartic acid and glutamic acid

Front 25

Give the formal definition of pH as an equation.

Back 25

pH=-Log(alphaH3O+)

Front 26

In the above equation, what does alpha H3O+ represent?

Back 26

Activity of the hydrated hydrogen ion

Front 27

Give the definition of Activity, as an equation.

Back 27

Activity=gamma[H3O+]

Front 28

What does gamma represent in the above equation?

Back 28

Gamma: activity co-efficient, giving a value of between 0-1

Front 29

Define equilibrium constant as an equation.

Back 29

Ka=[H3O+][A-]/[AH]

Front 30

Express the answer to the above flashcard logarithmically.

Back 30

pKa=-Log10Ka

Front 31

A lower pKa produces a higher Ka. What kind of acid is this?

Back 31

Stronger acid

Front 32

pKa of an acid is the pH when...?

Back 32

It is exactly half dissociated

Front 33

pH greater than the pKa, the acid is?

What about when the pH is less than the pKa?

Back 33

Acid is fully dissociated

Acid is predominantly HA

Front 34

The following equation may be derived from which equation (give its name):

pH=pKa+Log10([A-]/[HA])

Back 34

Henderson-Hasselbach Equation

Front 35

When [A-]=[HA] what does pH=?

Back 35

pKa

Front 36

Potential difference is generated between 2 electrodes immersed in solution. State the equation used to calculate this.

(Draw this out on paper)

Back 36

V=E^o+2.303RT/FxpH

Front 37

In the above equation, what does:

V, E^o, R, T and F signify?

Back 37

V: measured voltage

E^o: reference potential of the Ag/AgCl

R: gas constant J K^-1 M^-1

T: temperature K

F: Faraday's constant CM^-1

Front 38

Define the isoelectric point.

Back 38

pH at which the molecule contains no net electric charge

Front 39

Define a buffer.

Back 39

Resists a change in H+ concentration (pH) on addition of acid or alkali

Front 40

What identifies a good buffer on a Buffer Titration Curve?

Back 40

A flat plateau

Front 41

There are 6 properties to an ideal buffer. State 4 of them.

Back 41

Answer can include any 4 of the following:

>Impermeable to biological membranes

>Biological stability and lack of interferences 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

Front 42

Give an example of a Zwitterionic buffer.

Back 42

HEPES

Front 43

Seperation of proteins.

State 2 ways that mass can separate proteins.

State 2 ways that affinity can separate proteins.

Back 43

Centrifusion

Passage through membranes (dialysis)

Hydrophobic/hydrophilic property columns

Differential solubility extraction buffers

Front 44

State 3 ways that Charge and Mobility in Applied Fields can be used to seperate proteins.

Back 44

Buffer control

Separation on Gels

Electrophoresis