Coomassie Bluestained gel MW stds Cell extracts 33 44 78 18 132 Size kD Western blots take advantage of the sensitivity of antibodies to visualize proteins of interest in complex preparations ID: 226245
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Slide1
It's usually difficult to identify a protein of interest in a Commassie Blue-stained gel of cell extracts
Coomassie Blue-stained gel
MW stds.
Cell extracts
33
44
78
18
132
Size
(kD)
Western blots take advantage of the sensitivity of antibodies to visualize proteins of interest in complex preparationsSlide2
How are epitope tags used to locate proteins on western blots?
What steps are involved in western blots? Slide3
Proteins are expressed from plasmids that encode epitopes in the same reading frame with the cloned sequence
Protein of interest
Epitope added by plasmid sequences
Antibody that binds the epitope
Epitopes add antibody binding sites to proteinsSlide4
9 AA
ORF
His
6
HA
antibody binding domain of
S. aureus
protein A
14 AA
ORF
His
6
V5
pBG1805-encoded proteins
have multiple epitopes, which add 19 kDa to the protein’s MW
pYES2.1-encoded proteins
have two epitopes, which add ~5 kDa to the protein’s MW Slide5
Epitope-tagged proteins on gel
1. Primary antibody binds proteins (stoichiometric)
2. Secondary antibodies recognize multiple sites on primary antibody
3. Enzyme or fluorochrome amplifies the signal
Western blots
typically use multiple antibodies
to visualize epitope-tagged proteinsSlide6
HRP
HRP
Horseradish peroxidase (HRP)
40,000 MW enzyme
Catalyzes reaction that produces a colored product
Blue reaction product
3,3'5,5'-tetramethyl benzidine
+ H
2
O
2
HRP-catalyzed reaction amplifies the signal and generates a visible productSlide7
14 AA
ORF
His
6
V5
pYES2.1
9 AA
ORF
His
6
HA
IgG binding domain
pBG1805
Problem
: How can proteins encoded by pBG1805 and pYES2.1 plasmids be visualized on the same gel?
Sensitive (and expensive) monoclonal antibodies are available for the viral HA and V5 epitopes
The His
6 tag is useful for purifying proteins, but it is a poor antigenSlide8
Staphylococcus aureus
Protein A
A solution:
Use an alternative strategy to identify proteins encoded by pBG1805, based on the IgG binding domain of
S. aureus
protein A
Protein A is a transmembrane protein with multiple binding sites for the F
c
portion of IgGs
Helps bacterium to evade the host’s immune system during infectionsSlide9
Different strategies will be used to detect pBG1805- and pYES2.1-encoded proteins
pYES2.1 -encoded proteins on gel
Mouse monoclonal Ab detects V5 antigen
HRP
HRP
HRP
pBG1805-encoded proteins on gel
HRP
HRP
HRP-conjugated rabbit anti-mouse IgG polyclonal antibodySlide10
How are epitope tags used to locate proteins on western blots?
What steps are involved in western blots? Slide11
A multi-layer "sandwich" of filter papers and sponges encloses the gel and the PVDF membrane within a transfer cassetteSlide12
The cassette with its "sandwich" is inserted into the transfer apparatus
Be mindful of the correct polarity!
Black plate of sandwich toward black side of cassette holder
The electric current transfers proteins from the gel to the membraneSlide13
SDS-PAGE gel
PVDF membranes are hydrophobic. To bind proteins, they must first be wetted with methanol, followed by water and transfer buffer
Proteins are electrophoretically transferred to a PVDF (essentially
Teflon
) membraneSlide14
Blocking step
PVDF membranes will bind proteins nonspecifically. Milk contains high concentrations of casein proteins, which bind and saturate these low affinity, nonspecific sites on the membranes.
Epitope-tagged proteins (invisible)
MW standards
Membranes are incubated with 5% nonfat milk
MW standardsSlide15
Primary antibody binding step
Primary antibody is a mouse monoclonal antibody that recognizes the V5 epitope
1 2 3
1 – MW standards
2 – proteins with V5 epitope tag (pYES2.1)
3 – proteins with protein A domain (pBG1805)
Antibodies bind with high affinity to V5 epitopesSlide16
1 – MW standards2 – proteins with V5 epitope tag (pYES2.1)
3 – proteins with protein A domain (pBG1805)
Secondary antibody is a rabbit polyclonal antibody that:
recognizes the constant region of the mouse
IgGs
(lane 2)
binds to
S.
aureus
Protein A (lane 3)
1 2 3Slide17
1 – MW standards2 – proteins with V5 epitope tag (pYES2.1)
3 – proteins with protein A domain (pBG1805)
Detection
Blot is incubated with substrates for HRP
1 2 3
Blot is incubated until reaction products appearSlide18
Blue-black bands indicate the position of epitope-tagged proteins
Western blot done by BC studentsSlide19
Process begins with the electrophoretic transfer of proteins to a membrane
Blocking
Membrane replica
Primary antibody
Secondary antibody
Enzymatic detection
Final blot