W2 Biochemical Methods to Study Protein-protein Interactions

Question 1

difference between SDS PAGE and native PAGE

Answer 1

SDS: denaturing conditions > protein structure and protein interactions are lost; used to separate proteins in cell

native: non-denaturing conditions > protein structure remained; used for low complexity samples (purified proteins and complexes)

Question 2

purpose of adding glycerol into SDS-PAGE

Answer 2

makes sample “heavy” > can settle in the gel pocket

Question 3

difference between coomassie vs silver stain

Answer 3

silver staining x10 more sensitive

neither stain is quantitative and not all proteins are stained equally well

Question 4

the 4 biochemical methods to study protein-protein interactions

Answer 4

immunoprecipitations

affinity purification (AP) and tandem AP

GST pulldown assays

Yeast two hybrid (Y2H) system

Question 5

difference between IP and Co-IP

Answer 5

IP: isolation of a protein of interest from complex protein sample

Co-IP: isolation of a protein complex of interest from a complex protein sample

Question 6

what are nanobodies used in IP

Answer 6

recombinantly produced antigen binding VHH fragments derived from alpaca heavy chain IgG antibody > so that gel will only show one band

conventional antibody will give 3 bands (POI, heavy and light chain)

Question 7

process of tandem AP

Answer 7

two step purification method

attach 2 tags to POI > first affinity column using first tag > use TEV protease to cut off first tag > second affinity column > release second tag to give pure native protein complex

Question 8

how does GST pulldown assays work

Answer 8

GST fused to POI > expressed in cells and add glutathione agarose beads that bind specifically to GST > incubate with protein predicted to bind directly to POI > proteins eluted and carry out Western Blot > if band corresponding to protein predicted to bind to POI, means is direct interaction

Question 9

difference in principles of AP, IP and GST pulldown

Answer 9

AP: use of affinity matrices > mild elution releases native and intact protein complexes

IP: use of antibodies > elution under denaturing conditions

GST pulldown: use of purified proteins > elution under native or denaturing conditions

Question 10

how does Y2H system work

Answer 10

POI fused to DNA binding domain (DBD) of transcription factor and potential interacting protein fused to activation domain (AD)

if 2 proteins interact directly > DBD and AD in close proximity > reconstitute functional transcription factor > activate transcription at reporter gene

Question 11

different methods to reduce sample complexity

Answer 11

differential (velocity) centrifugation: separate organelles on basis of their sedimentation rate

density gradient centrifugation: separates organelles based on their sedimentation rate or density

Question 12

process of BioID-mediated biotin labeling

Answer 12

POI fused with BirA and incubated with biotin > BirA converts biotin into reactive form that can covalently attach to nearby lysine residues on proteins within 10nm > captured proteins analysed using MS to identify biotinylated proteins

Question 13

process of APEX-mediated biotin labelling

Answer 13

APEX fused into target protein > incubated with biotin phenol > cells treated with H2O2 > activates APEX > APEX catalyse oxidation of biotin-phenoxyl radical > rapidly labels nearby proteins by attaching biotin to their tyrosine residues > captured proteins analysed via MA