Practicals Flashcards
What is affinity chromatography used for?
Separating proteins based on reversible interaction between a protein and specific ligand attached to chromatographic matrix
How does affinity chromatography work?
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
What are some methods of performing desorption?
Performed specifically = using competitive ligands
Performed non-specifically = by changing pH, ionic strength or polarity
After affinity chromatography, describe the outcome of the sample
Protein is collected in purified and concentrated form
What are protein A and protein G?
Bacterial proteins
What do protein A and protein G bind to?
Bacterial proteins, which bind specifically to the Fc region of the polyclonal and monoclonal IgG-type Ab
What are protein A and protein G used for?***
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
Where are glycoproteins found?
Blood, secretions, cell membranes and connective tissue
What makes a glycoprotein?
Sugar/Carb moieties covalently linked to polypeptide backbone
Either linked via Asp or Ser/Thr
What was the glycoprotein stain used in this practical?
Periodic acid = oxidizing agent
Glycol oxidized into aldehydes
Shiff reagent reacts with aldehyde = magenta product formed
What enzyme is used for deglycosylation of N-linked glycans?
PNGase F
Glycosidase to remove the glycol moiety from protein of interest
What enzyme is used for deglycosylation of O-linked glycans?
Exoglycosidases = trim glycans to a short core
THEN
O-glycosidase = removes the glycan
What are the 3 steps of basic protein purificaation?
Equilibrate-Binding
Washing
Eluting
What buffers were used for protein purification and why?
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
Which protein beads did we use in this practical and why?
Protein G beads are specific for sheep polyclonal Ab
Used to trap the Ab on the sepharose matrix
Why do we neutralize the eluate of IP?
Because the elution is done with pH2.8 buffer
Neutralizing this prevents the protein from denaturing
How does ultrafiltration work?
Centrifuge sample through a semi-permeable membrane
To choose the correct ultrafiltration size, what do we have to consider?
Rotor and G force
Concentration factor
Adjust spinning time and molecular weight cut-off (MWCO)
What does ultrafiltration do?
Concentrate sample to desired volume
Remove unwanted small molecules (e.g salts and small proteins)
What was the role of fetuin in the practical?
Positive control for endoglycosidaze enzymes that cleave both N- and O-linked carbs
What conditions does PNgase F work under?
Digestion is performed under DENATURED conditions
How does Coomassie blue dye interact with the protein?
Binds primarily to Arg residues
