CPY Assay: Difference between revisions

Latest revision as of 19:57, 12 August 2010

Carboxypeptidase Secretion Assay

Revive Yeast Cells (2-3) days
Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast on selective plates, allow to grow for 1 day. (24°C)
Place nitrocellulose filters on gel and incubate overnight at 23°C. (Make sure to roll the sheet with a tube)
Wash filter in water.
Block filter with BSA for 1 hour.
Wash in primary antibody overnight at 4C or >3h at room temp

(10,000x dilution)

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 == Protocol ==
 *Revive Yeast Cells (2-3) days
-*Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast. (Does this have to be selective plates? or are YPD plates always used?)
+*Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast on selective plates, allow to grow for 1 day. (24°C)
-*Place nitrocellulose filters on gel and incubate overnight at 23(So you're not actually moving the patch to the filter, but just allowing it to touch?)
+*Place nitrocellulose filters on gel and incubate overnight at 23°C. (Make sure to roll the sheet with a tube)
-*Wash filter in water (by adding water to dish and rocking?)
+*Wash filter in water.
-*Primary antibody (polyclonal against cpy location?) how long?
+*Block filter with BSA for 1 hour.
-*Secondary antibody (where/what is it and how long?)
+*Wash in primary antibody overnight at 4C or >3h at room temp
-Wash with pbs in between?
+(10,000x dilution)
+*Rinse in Tbst.
-What is ECL?
+*Secondary antibody at 10 000X for 45min-1h.
+*Rinse in TBST.
+*Develop