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sec61-2 fusion proteins in yeast

[iso-8859-1] atish kumar mnlatish at yahoo.co.in
Mon May 13 08:56:58 EST 2002


Hello 
iam working on the sec61-2(mutated form of the yeast
translocan sec61) a substrate for the ER degradation
in yeast at first i fused the URA3 protein to the
sec61-2to its C terminus  but i failed to observe the
functional ura3 activity and i couldnt even  seen the
expression of myfusion protein  on a westernblot by
using  the antibodeies againast sec61 i expressed my
protein on a centromeric plasmid , later i fused the
leu2 to  sec61-2 on its C terminus now i can observe
the growth on a LEU negative media but i cant detect
the expression of my fusionprotein on a western blot
by using the sec61 antibodies but i can see the normal
sec61 which shows that western worked and the DNA
sequencing also shows to me that there are no
mutations and i can able to rescue my plasmid from
yeast showing its not rejected by yeast  it  is most
surprising to me , is there any special conditions
required to observe the fusion ER membrane proteins 
 like sec61 on a western blot , and also i fused URA3
to 2 other ER membrane proteins but iam not able to
getting  the functional URA3 activity eventhough i can
detect my fusion proteins on a western blot is it
necessary to follow certain conditions for fusing the
URA3 to ER membrane proteins to get functional URA3
activity i will be extremely thankful for kind
suggestions and comments
Thanking you
Atish kumar
Graduate student
centere for cellular and molecular biology
Hyderabad
India


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