1 reply to this topic

[biocrazy]

Dear Friends,

     I am here to seek your help regarding heterologous protein(from plant source) expression in Yeast. I used pYES-DEST52 vector (gateway cloning) and S.cerevisiae EGY48 competent cell. The protocol as follows

1. Inoculation in SD medium + 2 % Raffinose
2. ON, 200 rpm, 30 oC
3. Subculture into  SD medium + 2 % galactose (since vector is gal inducible system).
4. Collected 1 ml of culture from 0, 4, 8, 12, 16,20, 24 h
5. Isolated protein using the yeast protein extraction kit from GE healthcare.
but i could not see the expressed protein, even i could not find any signal even in western blot.

If anyone could help me then it will be very kind of you.

-Selvam Ayarpadikannan

[truta]

biocrazy, on 13 May 2011 - 05:05 AM, said:

Dear Friends,

     I am here to seek your help regarding heterologous protein(from plant source) expression in Yeast. I used pYES-DEST52 vector (gateway cloning) and S.cerevisiae EGY48 competent cell. The protocol as follows

1. Inoculation in SD medium + 2 % Raffinose
2. ON, 200 rpm, 30 oC
3. Subculture into  SD medium + 2 % galactose (since vector is gal inducible system).
4. Collected 1 ml of culture from 0, 4, 8, 12, 16,20, 24 h
5. Isolated protein using the yeast protein extraction kit from GE healthcare.
but i could not see the expressed protein, even i could not find any signal even in western blot.

If anyone could help me then it will be very kind of you.

-Selvam Ayarpadikannan

Hi!
Are you sure the plasmid is correct, i.e, right orientation relative to the GAL promoter?
How did you transform it into yeast?
Do you keep the selective pressure by always growing them in -URA media?