Practicals

Question 1

What is affinity chromatography used for?

Answer 1

Separating proteins based on reversible interaction between a protein and specific ligand attached to chromatographic matrix

Question 2

How does affinity chromatography work?

Answer 2

Target protein is specifically and reversibly bound by a complementary binding substance (ligand)

Specific binding occurs under favourable conditions = unbound material is washed away

Then target protein is recovered by changing conditions to favour desorption

Question 3

What are some methods of performing desorption?

Answer 3

Performed specifically = using competitive ligands

Performed non-specifically = by changing pH, ionic strength or polarity

Question 4

After affinity chromatography, describe the outcome of the sample

Answer 4

Protein is collected in purified and concentrated form

Question 5

What are protein A and protein G?

Answer 5

Bacterial proteins

Question 6

What do protein A and protein G bind to?

Answer 6

Bacterial proteins, which bind specifically to the Fc region of the polyclonal and monoclonal IgG-type Ab

Question 7

What are protein A and protein G used for?***

Answer 7

When immobilized, they are high capacity adsorbents for many routine applications

Examples
Purification of monoclonal IgG type Ab
Isolation of polyclonal IgG and its subclasses and purification of immune complexes

Question 8

Where are glycoproteins found?

Answer 8

Blood, secretions, cell membranes and connective tissue

Question 9

What makes a glycoprotein?

Answer 9

Sugar/Carb moieties covalently linked to polypeptide backbone

Either linked via Asp or Ser/Thr

Question 10

What was the glycoprotein stain used in this practical?

Answer 10

Periodic acid = oxidizing agent
Glycol oxidized into aldehydes

Shiff reagent reacts with aldehyde = magenta product formed

Question 11

What enzyme is used for deglycosylation of N-linked glycans?

Answer 11

PNGase F

Glycosidase to remove the glycol moiety from protein of interest

Question 12

What enzyme is used for deglycosylation of O-linked glycans?

Answer 12

Exoglycosidases = trim glycans to a short core
THEN
O-glycosidase = removes the glycan

Question 13

What are the 3 steps of basic protein purificaation?

Answer 13

Equilibrate-Binding

Washing

Eluting

Question 14

What buffers were used for protein purification and why?

Answer 14

BINDING & WASHING
Buffer A = promotes binding of albumin to affi-gel blue by creating lower neutral pH

ELUTION
Buffer B promotes desorption of albumin from affi-gel blue because of higher ionic/salt strength

Question 15

Which protein beads did we use in this practical and why?

Answer 15

Protein G beads are specific for sheep polyclonal Ab

Used to trap the Ab on the sepharose matrix

Question 16

Why do we neutralize the eluate of IP?

Answer 16

Because the elution is done with pH2.8 buffer

Neutralizing this prevents the protein from denaturing

Question 17

How does ultrafiltration work?

Answer 17

Centrifuge sample through a semi-permeable membrane

Question 18

To choose the correct ultrafiltration size, what do we have to consider?

Answer 18

Rotor and G force

Concentration factor

Adjust spinning time and molecular weight cut-off (MWCO)

Question 19

What does ultrafiltration do?

Answer 19

Concentrate sample to desired volume

Remove unwanted small molecules (e.g salts and small proteins)

Question 20

What was the role of fetuin in the practical?

Answer 20

Positive control for endoglycosidaze enzymes that cleave both N- and O-linked carbs

Question 21

What conditions does PNgase F work under?

Answer 21

Digestion is performed under DENATURED conditions

Question 22

How does Coomassie blue dye interact with the protein?

Answer 22

Binds primarily to Arg residues