BIOB10H3 Lecture Notes - Lecture 9: Photographic Film, Cell Membrane, Knockout Mouse
25 Jun 2018
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Lecture 9: Studying Proteins
Studying Proteins
Determining primary structure
Investigating secondary/tertiary structure
Investigating protein localization
Investigating protein function
many of these techniques involve: protein purification
Studying Proteins – Primary Structure
1. Extrapolate from genomic sequence
2. Manually sequence the primary structure
this requires protein purification
Genomic Sequence - Extrapolate amino acid sequence from gene/nucleotide sequence from computer
databases of genomes
1980’s and 1990’s - “age of genomics”
The Human Genome Project vs. Celera
DNA sequences now in Genbank
Craig Ventor - founded Celera Genomics in 1999
Extrapolating from Genomic Sequence
This has 2 limitations/issues:
1. Introns/exons
exons: code for proteins
introns: intervening sequences
- don’t code for anything
- spliced out of mRNA
- will not correctly predict protein sequence
2. Posttranslational modifications often cannot be predicted from genome
proteolysis
glycosylation
Manually Sequence the Primary Structure
“The age of proteomics”
Mass spectrometry
osequencing the primary sequence of proteins
o*First*- purify proteins:
proteins either separated from a complex protein mixture on gel
or proteins purified using chromatography
find more resources at oneclass.com
find more resources at oneclass.com
Purifying
Proteins
Protein Gel Electrophoresis
ocell lysates are made from cells of interest
oproteins are denatured by heat and put in tracking dye containing SDS
SDS is negatively charged - coats proteins and makes all proteins negatively charged
oproteins are separated on a gel based on size
oGel - polyacrylamide gel
porous gel with many holes
smaller proteins (low kDa) will move through quickly and larger proteins (high kDa) will
move through very slowly
oproteins move through by electrophoresis
an electrical current is applied through the gel so there is a positively charged end
(anode) and a negatively charged end (cathode)
proteins are all negative, so they will move towards the anode
othis is 1D gel electrophoresis ------------------>
ofor 2D gel electrophoresis - proteins are first ----------->
separated (Isoelectric focusing)
based on the charge of the proteins
1000’s of proteins can be resolved using 2D gel
electrophoresis
Chromatography
oto isolate proteins from cell lysates
olysates poured into column packed with beads
find more resources at oneclass.com
find more resources at oneclass.com
o3 major kinds of chromatography:
gel-filtration chromatography
- separates proteins based on size
- beads have different pore sizes
- similar concept to gel
electrophoresis
ion-exchange chromatography
- separates proteins based on charge
- beads have a charge
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Many of these techniques involve: protein purification. Studying proteins primary structure: extrapolate from genomic sequence, manually sequence the primary structure this requires protein purification. Genomic sequence - extrapolate amino acid sequence from gene/nucleotide sequence from computer databases of genomes. Craig ventor - founded celera genomics in 1999. Introns/exons exons: code for proteins introns: intervening sequences don"t code for anything spliced out of mrna. Will not correctly predict protein sequence: posttranslational modifications often cannot be predicted from genome. Mass spectrometry: sequencing the primary sequence of proteins, *first*- purify proteins: Proteins either separated from a complex protein mixture on gel. Purifying: cell lysates are made from cells of interest, proteins are denatured by heat and put in tracking dye containing sds. Sds is negatively charged - coats proteins and makes all proteins negatively charged: proteins are separated on a gel based on size, gel - polyacrylamide gel.
