It's usually difficult to identify a protein of interest in a Commassie Blue-stained gel of cell...
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Transcript of It's usually difficult to identify a protein of interest in a Commassie Blue-stained gel of cell...
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 preparations
How are epitope tags used to locate proteins on western blots?
What steps are involved in western blots?
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 proteins
9 AA
ORFHis6
HA antibody binding domain of S. aureus protein A
14 AA
ORFHis6V5
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
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 proteins
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
+ H2O2
HRP-catalyzed reaction amplifies the signal and generates a visible product
14 AA
ORF His6V5
pYES2.1
9 AA
ORFHis6 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 His6 tag is useful for purifying proteins, but it is a poor antigen
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 Fc portion of IgGs
Helps bacterium to evade the host’s immune system during infections
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 antibody
How are epitope tags used to locate proteins on western blots?
What steps are involved in western blots?
A multi-layer "sandwich" of filter papers and sponges encloses the gel and the PVDF membrane within a transfer cassette
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 membrane
SDS-PAGE gelPVDF 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) membrane
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 standards
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 epitopes
1 – MW standards
2 – 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 3
1 – MW standards
2 – 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 appear
Blue-black bands indicate the position of epitope-tagged proteins
Western blot done by BC students
Process begins with the electrophoretic transfer of proteins to a membrane
BlockingMembrane replica Primary antibody
Secondary antibody Enzymatic detection Final blot