extraction of extracellular protein of C. neoformans - extraction method (Jun/12/2005 )
hi,
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
-Chan Mun Yee-
QUOTE (Chan Mun Yee @ Jun 12 2005, 06:38 PM)
hi,
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
Hi Chan,
I never extract protein using the kit that you mention. Could you please tell me how you generate your three fractions?
Regard
Hadrian
-Hadrian-
QUOTE (Hadrian @ Jun 15 2005, 08:45 AM)
QUOTE (Chan Mun Yee @ Jun 12 2005, 06:38 PM)
hi,
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
Hi Chan,
I never extract protein using the kit that you mention. Could you please tell me how you generate your three fractions?
Regard
Hadrian
-Chan Mun Yee-
QUOTE (Chan Mun Yee @ Jun 21 2005, 07:18 PM)
QUOTE (Hadrian @ Jun 15 2005, 08:45 AM)
QUOTE (Chan Mun Yee @ Jun 12 2005, 06:38 PM)
hi,
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
1)does anyone here tried to extract C. neoformans using the partial yeast extraction kit (P-PEK by Calbiochem)? I have generated all the three fractions, however i could not get any band with the SDS-PAGE. this has prevented me from proceeding with the 2DE.
2) I'm having problems regarding the proper way to extract extracellular protein of C. neoformans. I have tried to grow it in 200ml of chemically defined media for 1 week, precipitate it with 10% TCA, wash with ice-chilled acetone but the concentration of the protein was far too low for 2DE. by the way,what is the proper volume of yeast peptone dextrose used to culture extracellular proteins of C. neoformans for 2DE? anyone with advice on other protocols?
a million thanks ahead.........
mun yee
Hi Chan,
I never extract protein using the kit that you mention. Could you please tell me how you generate your three fractions?
Regard
Hadrian
Hi Hadrian,
Basically the kit allow u 2 prepare a serial of sample preparation of complex protein mixtures using reagent mixtures with increasing solubilization strength.
1)Extraction reagent 1-the most soluble protein of yeast cells (Fraction 1)
2) Extraction reagent 3- for solubilization of proteins otherwisw insoluble in Extraction reagent 1, efficient for membrane protein extraction. (Fraction 2)
3) SDS-buffer- for proteins still insoluble in Extraction reagent 2 (Fraction 3)
Hope this helps.
Chan
-Chan Mun Yee-