Difference between revisions of "CPY Assay"
From Bridges Lab Protocols
(Created page with 'Carboxypeptidase Secretion Assay == 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 sele...') |
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== Protocol == | == Protocol == | ||
*Revive Yeast Cells (2-3) days | *Revive Yeast Cells (2-3) days | ||
| − | *Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast | + | *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 | + | *Place nitrocellulose filters on gel and incubate overnight at 23°C. (Make sure to roll the sheet with a tube) |
| − | *Wash filter in water | + | *Wash filter in water. |
| − | * | + | *Block filter with BSA for 1 hour. |
| − | *Secondary antibody | + | *Wash in primary antibody overnight at 4C or >3h at room temp |
| + | (10,000x dilution) | ||
| + | *Rinse in Tbst. | ||
| + | *Secondary antibody at 10 000X for 45min-1h. | ||
| + | *Rinse in TBST. | ||
| + | *Develop | ||
Latest revision as of 19:57, 12 August 2010
Carboxypeptidase Secretion Assay
Protocol
- 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)
- Rinse in Tbst.
- Secondary antibody at 10 000X for 45min-1h.
- Rinse in TBST.
- Develop
