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JOHANN PETER HOSSLE
14. Non-adsorbed proteins are removed by three successive washing cycles, starting with washing buffer (pH 4.0), then switching to pH 8.0
and a final wash at pH 4.0. Before and after this treatment and also
in between the individual washing steps the material is recovered by
centrifugation at 2'500 x g for 5 minutes at 4°C.
15. The Sepharose-coupled bacteriallysate can be storedas a suspension in
PBS (+0.01% thimerosal) at 4°C for several weeks.
Preadsorbtion of the antibodies used for screening
1. The undiluted primary antibody as well as the secondary antibody ( e.g.
peroxidase-coupled goat anti-rabbit, Calbiochem) at a protein concentration of 0.5 to 1 mg/ml are each mixed with 1.7 volumes of Sepharosecoupled bacteriallysate and incubated overnight at 4°C on a rotating
wheel. Foreachscreening cycle approx. 300 ~-tl of treated primary antibody (final working dilution: 1:100) and 15~-tl oftreated secondary antibody (final working dilution: 1:2000) are required.
2. The suspensions are filled into separate pipette tips that are plugged with
a small amount of glass wool. Non-retarded antihoclies are eluted with
cold blocking buffer (at least 5 x times the column volume).
3. The concentration of the eluted antibody solutions is adjusted with cold
blocking buffer to 1/20 of the original concentration. Thimerosal is
added (0.01% final).
Note: Thimerosal is used instead ofNa-azide, if mold contamination of peroxidase-conjugated antibody solutions is tobe prevented. Antibody solutions are now ready to use and can be stored at 4°C.
Plating
1. Plating is performed as described above, with the following changes:
a. One additional 12 ml screw-cap tube containing 2.5 ml top-agar is
placed in the water bath at 49°C for plating of an induction control.
b. In one infection mix, 100 ~-tl of a phage suspension containing a known
percentage of wild-type phage with an intact ß-galactosidase gene is used
to serve as an induction control.
c. For platingofthe induction control20 ~-tlX-Gal is added to the 2.5 ml of
top-agar immediately before adding to the corresponding infection mix.
JOHANN PETER HOSSLE
14. Non-adsorbed proteins are removed by three successive washing cycles, starting with washing buffer (pH 4.0), then switching to pH 8.0
and a final wash at pH 4.0. Before and after this treatment and also
in between the individual washing steps the material is recovered by
centrifugation at 2'500 x g for 5 minutes at 4°C.
15. The Sepharose-coupled bacteriallysate can be storedas a suspension in
PBS (+0.01% thimerosal) at 4°C for several weeks.
Preadsorbtion of the antibodies used for screening
1. The undiluted primary antibody as well as the secondary antibody ( e.g.
peroxidase-coupled goat anti-rabbit, Calbiochem) at a protein concentration of 0.5 to 1 mg/ml are each mixed with 1.7 volumes of Sepharosecoupled bacteriallysate and incubated overnight at 4°C on a rotating
wheel. Foreachscreening cycle approx. 300 ~-tl of treated primary antibody (final working dilution: 1:100) and 15~-tl oftreated secondary antibody (final working dilution: 1:2000) are required.
2. The suspensions are filled into separate pipette tips that are plugged with
a small amount of glass wool. Non-retarded antihoclies are eluted with
cold blocking buffer (at least 5 x times the column volume).
3. The concentration of the eluted antibody solutions is adjusted with cold
blocking buffer to 1/20 of the original concentration. Thimerosal is
added (0.01% final).
Note: Thimerosal is used instead ofNa-azide, if mold contamination of peroxidase-conjugated antibody solutions is tobe prevented. Antibody solutions are now ready to use and can be stored at 4°C.
Plating
1. Plating is performed as described above, with the following changes:
a. One additional 12 ml screw-cap tube containing 2.5 ml top-agar is
placed in the water bath at 49°C for plating of an induction control.
b. In one infection mix, 100 ~-tl of a phage suspension containing a known
percentage of wild-type phage with an intact ß-galactosidase gene is used
to serve as an induction control.
c. For platingofthe induction control20 ~-tlX-Gal is added to the 2.5 ml of
top-agar immediately before adding to the corresponding infection mix.