Difference between revisions of "CPY Assay"

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(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. (Does this have to be selective plates? or are YPD plates always used?)  
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*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?)
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*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?)
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*Wash filter in water.
*Primary antibody (polyclonal against cpy location?) how long?
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*Block filter with BSA for 1 hour.
*Secondary antibody (where/what is it and how long?)
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*Wash in primary antibody overnight at 4C or >3h at room temp
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(10,000x dilution)  
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*Rinse in Tbst.
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*Secondary antibody at 10 000X for 45min-1h.
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*Rinse in TBST.
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*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