304
JOHANN PETER HOSSLE
Secondary antibody incubation
Color development for
peroxidase
conjugated
secondary
antibodies
room temperature for at least 2 hrs. To ensure that the filters remain
completely soaked with antibody solution, the petri dishes are placed
on a rocking platform during incubation.
4. Unbound primary antibodies are removed bywashing the membranes (5
at a time) for 3 x 10 minutes in 25 ml TBT at room temperature while
shaking.
5. This step is performed as in step 3. The working dilution of the preadsorbed secondary antibody typically is approx. 1:2000 in 3 ml blocking
buffer (e.g. peroxidase-coupled goat anti-rabbit, Calbiochem). Secondary antibody working solutions should not be reused more than once.
After the second usage, the capacity of the secondary antibody working
solution seems to be exhausted and often results in a sudden loss of positive signals.
6. Unbound secondary antibodies are removed bywashing the membranes
3 x 10 minutes in 25 ml TBS at room temperature while shaking.
7. In a plastictray 45 ml cold ( 4 oC) enzyme reaction bufferare mixed with 5 ml
cold ( -20°C) chloronaphthol and 50 J.ll H20 2• A maximum of 5 membrane
filters at a time are placed into this color development solution and gently
agitated. Within several minutes positive purple plaques should develop.
8. Nitrocellulose filters are placed on a light box, if results are documented
by Polaroid photography (film type 665; f-stop 4.5; shutter speed 1/15;
orange filter).
9. To assist subsequent picking of positive plaques, it is convenient to copy
the pattern of positive plaques with the corresponding needle stick marks
onto a transparent overhead film.
Plaque purification
1. Areas corresponding to positive plaque signals are punched out of the
top-agar using the wide end of a sterile Pasteur pipette. The thin top-agar
disk usually comes easily off the plate and can be suspended into 0.5 ml of
A.-dil in an eppendorf tube.
2. Phage particle contained in the punched-out plaques are released from
top-agar by vigorous vortexing for at least 1 min. The newly created
phage suspension is placed for at least 1 hr at room temperature before
plating again.
JOHANN PETER HOSSLE
Secondary antibody incubation
Color development for
peroxidase
conjugated
secondary
antibodies
room temperature for at least 2 hrs. To ensure that the filters remain
completely soaked with antibody solution, the petri dishes are placed
on a rocking platform during incubation.
4. Unbound primary antibodies are removed bywashing the membranes (5
at a time) for 3 x 10 minutes in 25 ml TBT at room temperature while
shaking.
5. This step is performed as in step 3. The working dilution of the preadsorbed secondary antibody typically is approx. 1:2000 in 3 ml blocking
buffer (e.g. peroxidase-coupled goat anti-rabbit, Calbiochem). Secondary antibody working solutions should not be reused more than once.
After the second usage, the capacity of the secondary antibody working
solution seems to be exhausted and often results in a sudden loss of positive signals.
6. Unbound secondary antibodies are removed bywashing the membranes
3 x 10 minutes in 25 ml TBS at room temperature while shaking.
7. In a plastictray 45 ml cold ( 4 oC) enzyme reaction bufferare mixed with 5 ml
cold ( -20°C) chloronaphthol and 50 J.ll H20 2• A maximum of 5 membrane
filters at a time are placed into this color development solution and gently
agitated. Within several minutes positive purple plaques should develop.
8. Nitrocellulose filters are placed on a light box, if results are documented
by Polaroid photography (film type 665; f-stop 4.5; shutter speed 1/15;
orange filter).
9. To assist subsequent picking of positive plaques, it is convenient to copy
the pattern of positive plaques with the corresponding needle stick marks
onto a transparent overhead film.
Plaque purification
1. Areas corresponding to positive plaque signals are punched out of the
top-agar using the wide end of a sterile Pasteur pipette. The thin top-agar
disk usually comes easily off the plate and can be suspended into 0.5 ml of
A.-dil in an eppendorf tube.
2. Phage particle contained in the punched-out plaques are released from
top-agar by vigorous vortexing for at least 1 min. The newly created
phage suspension is placed for at least 1 hr at room temperature before
plating again.