Immunoblot Screening of Chlorella Virus Plaques
Take the desired petri plates of virus plaques on chlorella lawns and chill in the cold room for 30-60 min prior to use.
Overlay the plates with the nitrocellulose circles.
Allow the nitrocellulose to lay on the agar surface for 15 min.
Allow the filters to air dry.
Add 7.5-8.0 mL of the blocking solution to each filter in a glass petri plate and incubate for 30 min with gentle shaking (approximately 100 rpm on the rotary shaker).
Decant the blocking solution from the petri plates.
Add 7.5-8.0 mL of a 1/600 dilution of the 1° antiserum in 1X TBST to each filter and incubate for 30 min, with gentle shaking.
Decant the antiserum from the petri plates.
Wash each filter 3X with 8.0 mL of 1X TBST for 10 min, with gentle shaking.
Decant the TBST solution from the petri plates.
Add 7.5-8.0 ml of a 1/7500 dilution of the 2° antiserum in 1X TBST to each of the filters and incubate for 30 min, with gentle shaking.
Decant the antiserum solution from the petri plates.
Wash each filter 3X with 8.0 mL of 1X TBST for 10 min, with gentle shaking.
Decant the TBST solution from the petri plates.
Add 7.5-8.0 mL of the reaction mixture to each of the filters, cover with aluminum foil and incubate with gentle shaking.
When the color development is sufficient, decant the reaction mixture from the petri plates.
Add 8.0 mL of the stop/storage buffer to each filter and incubate for 30 min, with gentle shaking.
Place the filters into a glass pyrex baking dish with the stop/storage buffer and incubate for 30 min at room temperature.
Remove the filters from the stop/storage buffer and allow to air dry on paper towels or filter papers.
Store the filters in small plastic sealed bags.
Place in the cold room overnight.
