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 .
