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Efficient lysis of E.coli cells

Lysozyme

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4 replies to this topic

#1 neuron

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Posted 06 April 2015 - 10:13 PM

Hi,

 

We are trying to lyse e.coli cells wherein we don't want to use sonicator and we don't have french press in lab. We want the protein complexes to be intact after lysis. So we tried adding lysozyme and perform lysis. But there are two problems-

 

1. How do we estimate the total protein content? If the answer is minus the added lysozyme then I have doubt. I found a paper where authers mentioned that lysozyme makes some protein complex in the membrane, so we don't know how much lysozyme we get in the lysate. 

 

2. We are getting very less efficiency i.e.after using 6gm pellet we get only 2ug/ul lysatesad.png . Which is not enough for our down stream applications

 

Are you aware of any other method of lysis through which we can get good yield of protein but intact complexes?

 

Please suggest.

 

Thanks



#2 Curtis

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Posted 06 April 2015 - 10:18 PM

What is the following experiment you want to do? why are you lysing E.coli? 



#3 neuron

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Posted 06 April 2015 - 10:43 PM

We want to do microscopy and purify a complex that binds to our molecule when we add that in culture. So we can't use sonicator 



#4 CandyTon

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Posted 15 April 2015 - 01:33 AM

This may help. http://www.creative-biolabs.com/bacterial-expression-systems-e-coli-bacillus.html. Just for a suggetion. smile.png



#5 phage434

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Posted 16 April 2015 - 04:49 PM

You may find that multiple freeze-thaw with liquid nitrogen and warm water would break open your cells quite effectively. EDTA in the buffer, and some protease inhibitors would likely be a good idea.






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