Lec 3-4 Flashcards
Why do we study protein function?
- knockdown or gene knockout
- transfection/ transformation overexpression studies
- protein localization w/i cells
- determine protein structure
- in vitro function
- identify interacting proteins
describe protein separation by SDS polyacrylamide gel electrophoresis
- proteins are boiled in strong SDS detergent to denature into the primary structure and make them negatively charged
- run the sample in gel, electrical current forces proteins to run downwards to a positive pole, separated by charge
- add coomassie blue to stain
- to detect specific proteins, can do western blotting if you have antibodies against the protein of interest, or if unknown, the protein can be identified by mass spectroscopy
what is the old way to purify proteins?
cell fractionation combined with vitro assay
what is immunoprecipitation?
a rapid form of affinity chromatography purification method
describe the steps of immunoprecipitation
- grind up cells in extraction buffer then centrifuge to obtain cell extract
- add an antibody against a specific protein, needs to bind at a high affinity
- add agarose beads coupled to a specific protein, then add detergent to elute everything else
- centrifuge and keep the pellet that contains beads-antibody-specific protein
how to purify protein without antibodies against it
clone the gene and express it in e.coli, can make lots of copies (overexpression)
how to obtain purified protein after expressing in e.coli
clone the gene into a vector with an epitope tag or affinity tag, express the fusion protein in bacteria or in vitro, then use IP to purify
what is an epitope tag?
a small protein sequence (10 a.a. long) that’s recognized by commercially available antibodies ex. HA tag
what is an affinity tag?
larger than an epitope tag, it’s a protein that can be easily purified based on its affinity for a specific substrate e.g. the GST tag has a specific binding affinity for glutathione
why do we express the tagged form of protein in bacteria and then purify it?
- use in vitro assays
- test for direct interactions b/w 2 purified proteins (GST pulldown)
- determine protein structure
- antibody production (study protein localization and perform co-IP to identify interacting proteins)
what is a fusion protein?
a protein fused with an epitope tag or affinity tag
how to make a fusion protein?
- use cDNA to amplify your gene by PCR - PCR primers also carry restriction site sequences for cloning
- clone your gene into a vector that already has an epitope tag or affinity tag in it (or for epitope tags, one of the primers contains an epitope sequence)
how to use PCR to insert an epitope tag?
PCR primers have restriction enzyme sites at ends that can clone the PCR product into a plasmid vector and 1 of the primers also has a sequence for an epitope tag ex. EcoR1 has HA
how do you make a fusion protein with a plasmid that already has an epitope tag?
clone into a GST (glutathione S-transferase) vector to express GST fusion protein
describe GST affinity purification
- clone gene of interest into GST vector, now express GST-protein x (gene of interest)
- bind GST-protein x to GSH (glutathione) beads, then mix and incubate
- wash beads, elute with GSH
- run eluate (exiting mixture) on protein gel
- now have purified GST-protein x
what is a GST pulldown?
tests for in vitro interaction between 2 known and cloned proteins
1. GST-protein x
2. HA-protein Y
what are the steps of a GST pulldown?
- bind GST-protein x to GSH beads
- incubate with HA-protein y
- wash beads, eluate with GSH
- run eluate on protein gel
if protein y can bind to protein x, it will stick to the beads so if we wash them multiple times then it should stay attached
describe protein expression in animal cells
- can express tagged form of protein in cultured cells or in whole organism
i) vector contains a strong inducible promoter for animal cell expression
ii) transfect into an animal to get transgenic animal
iii) like bacterial expression, the gene is cloned with an epitope tag or affinity tag or with GFP
iv) purify protein using epitope tag or affinity tag
v) directly study protein localization
vi) directly perform co-IP to identify interacting proteins
describe co-IP
- grind up cells in mild extraction buffer to preserve protein-protein interactions, centrifuge to obtain cell extract
- add an antibody against the specific protein
- add beads coupled to the specific protein
- centrifuge pellet which contains beads-antibody-specific protein
- known associated proteins can be identified by western blotting and unknown associated proteins can be identified by mass spec
what is mass spectrometry
a highly sensitive method for identifying unknown proteins, after co-IP the eluate is run on the SDS page, then stained with coomassie, and the prominent bands are cut out THEN the proteins are broken into peptides and ionized which determines the mass/charge ratio and software determine what combo of a.a. can give that mass/charge ratio and then you can search protein databases for predicted proteins with those a.a.
what can you use to test interactions between 2 known proteins?
- GST pulldown (can they interact)
- CoIP, then Western (do they interact)
what can you use to identify unknown interacting proteins?
CoIP, then mass spec (what interacts)
what does BLASTp help identify?
related proteins in the same or other species (identifies homologues, paralgues)
what does prosite help identify?
putative domains based on sequence similarity to characterized domains (possible functions, indicate relationship to known gene families)
