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