Find content
Not Telling
TQ
Really need help!
Topics I've Started
Plasmid isolation from agrobacterium
03 February 2013 - 10:43 PM
Hi, anyone here knows the protocol to isolate the binary vector from agrobacterium?
TQ
Really need help!
TQ
Really need help!
Adh1
22 January 2013 - 12:33 AM
Hi,
Anyone here work with Adh1 from eukaryotes?
where is Adh1 actually secreted when it is expressed in E.coli?
need help!
Anyone here work with Adh1 from eukaryotes?
where is Adh1 actually secreted when it is expressed in E.coli?
need help!
problem in extracting the protein
22 January 2013 - 12:18 AM
Hi.
Before I ask, i want to explain one by one so that people that reading my post will not get confuse
i'm working with Adh1 from eukaryotes, and i expressed it in BL21 (DE3).
I think BL21 (DE3) had successfully expressed my gene because there are extra band on SDS-PAGE (my control is BL21 carrying pET-41a(+)).
I extract the total protein by using sample buffer that contain SDS, Tris-HCl, glycerol, deionized water. after I pelleted the cell, i will resuspended it with sample buffer, heat at 95 degree and vortex it vigorously.
my wonder now is, although I treat it "harsh", seems like the SDS did not lysed my cell completely. I try to add some more of sample buffer, but still...the cell extract looked cloudy. If the lysis is complete, the cell extract should be look translucent right?-correct me if i'm wrong.
anyone can explain why this is happened?
Before I ask, i want to explain one by one so that people that reading my post will not get confuse
i'm working with Adh1 from eukaryotes, and i expressed it in BL21 (DE3).
I think BL21 (DE3) had successfully expressed my gene because there are extra band on SDS-PAGE (my control is BL21 carrying pET-41a(+)).
I extract the total protein by using sample buffer that contain SDS, Tris-HCl, glycerol, deionized water. after I pelleted the cell, i will resuspended it with sample buffer, heat at 95 degree and vortex it vigorously.
my wonder now is, although I treat it "harsh", seems like the SDS did not lysed my cell completely. I try to add some more of sample buffer, but still...the cell extract looked cloudy. If the lysis is complete, the cell extract should be look translucent right?-correct me if i'm wrong.
anyone can explain why this is happened?
