lab techniques 2

Question 1

differential centrifugation

Answer 1

• separating cell compartments by density
• used to determine cellular/subcellular location

Question 2

immunofluorescence

Answer 2

• used to determine cellular/subcellular location
• use labeled antibody to detect
• primary antibody attaches to protein and secondary antibody that is labeled with fluorescent dye
• must kill cells for intracellular staining

Question 3

how can fusion proteins be used to determine cellular location

Answer 3

• useful if the protein of interest doesn’t have an antibody
• make a reporter construct that is fusion of protein of interest and reporter protein (like GFP)
• sequence on some kind of expression contruct such as a plasmid or viral vector

Question 4

what role do epitope tags play in protein fusion

Answer 4

• useful if you dont have an antibody for the protein of interest
• make fusion of protein of interest with epitope tag
• epitope tag is a sequence that is bound to an antibody
• antibody can be attached to a fluorescent molecule

Question 5

affinity chromatography

Answer 5

• use antibody against one protein to capture interacting proteins

Question 6

co-immunoprecipitation

Answer 6

• substrate is incubated in a tube with antibody-coupled resin
• other molecules are spun and washed off
• remaining materials are spun and antigen and protein interacting with antigen are eluted
• proteins analyzed using SDS-PAGE and western blot

Question 7

how would you accomplish co-IP or affinity chromatography without an antibody for the protein of interest

Answer 7

• GST pull-down
• make a fusion protein with the protein of interest and a protein or aa sequence with a high affinity ligand as “bait”
• target interacting protein is prey

Question 8

FRET

Answer 8

• fluorescence resonance energy transfer
• uses two fusion proteins with protein of interest and fluorescent protein
• introduce constructs to the cells
• excite at one wavelength and detect emission wavelength
• absorption spectrum of acceptor molecule must overlap with the emission of the donor molecule
• more effective at shorter distances (within 50-100 angstroms)

Question 9

Yeast two-hybrid

Answer 9

• way of testing protein-protein interactions
• requires DNA binding domain (DBD) and transcriptional activation domain (AD)
• DBD is on the bait vector and AD is on the prey
• DBD binds to DNA and if the two proteins interact, AD will also bind and recruit RNA polymerase and reporter gene will be expressed
• if the proteins don’t interact, the reporter gene will not be expressed

Question 10

how would you use yeast two-hybrid if you dont have a hypothesis about which proteins will interact

Answer 10

• add DBD and bait, lac Z as reporter gene and mRNA for different potential prey proteins with the prey protein
• if no interaction, no lacZ expression, white colonies
• if interaction - activation of transcription, blue colonies will grow
• collect and sequence blue colonies to identify interacting protein

Question 11

what are some pros and cons of yeast 2 hybrid

Answer 11

pros:
- relatively simple
- easy to sequence plasmid DNA to determine interacting proteins
cons:
- proteins being tested may require factors absent in yeast, like chaperones
- fusion to DBD or AD may interfere with binding regions of proteins