Difference between revisions of "CPY Assay"
From Bridges Lab Protocols
(→Protocol) |
|||
| Line 4: | Line 4: | ||
*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 on selective plates, allow to grow for 1 day. (24°C) | *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. | + | *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. | ||
*Wash in primary antibody overnight at 4C or >3h at room temp | *Wash in primary antibody overnight at 4C or >3h at room temp | ||
| + | (10,000x dilution) | ||
*Rinse in Tbst. | *Rinse in Tbst. | ||
*Secondary antibody at 10 000X for 45min-1h. | *Secondary antibody at 10 000X for 45min-1h. | ||
*Rinse in TBST. | *Rinse in TBST. | ||
*Develop | *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
