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problem in extracting the protein

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#1 An-nur Al-Irsyad

An-nur Al-Irsyad

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Posted 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 Posted Image

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?

#2 phage434

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Posted 22 January 2013 - 05:56 AM

I would not describe it as translucent. It still looks as if it has lots of junk in it, but not as much. It should be high viscosity before you pellet it, due to released DNA -- a gloppy mess. After vortexing and pelleting, it should be much lower viscosity. This may be difficult to achieve with just vortexing, and may require sonication or treatment with a DNAse, such as benzonase.





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