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Mammalian Cell Lysis Buffer Triton-X100 Content

cell lysis buffer

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6 replies to this topic

#1 alphawhiskey

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Posted 07 January 2014 - 06:42 PM

Hi all,

 

I am trying to lyse COS-7 cells to detect actin-related proteins in the cytoplasm. My original lysis buffer recipe is:

 

150mM NaCl

1% Triton-X

50mM Tris pH 8.0

Benzamidine HCl 1mM

 

However, because the Triton-X reacts with Bradford, should I reduce it to 0.05% Triton-X? Would that be enough to lyse mammalian cells?

 

 



#2 bob1

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Posted 07 January 2014 - 06:47 PM

That should be enough to put holes in the membranes, but it may not completely lyse the cells.



#3 alphawhiskey

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Posted 08 January 2014 - 10:17 PM

Would you recommend sonication or passing the cells through syringe and needle?



#4 bob1

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Posted 08 January 2014 - 11:17 PM

Possibly, but it depends on your final application - sonication may well disrupt interactions, so IP may not be possible.  Syringe and needle is gentler.  You could also use a homogenizer such as a Potter-Elvehejm.



#5 memari

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Posted 09 January 2014 - 12:40 PM

Hi all,

 

I am trying to lyse COS-7 cells to detect actin-related proteins in the cytoplasm. My original lysis buffer recipe is:

 

150mM NaCl

1% Triton-X

50mM Tris pH 8.0

Benzamidine HCl 1mM

 

However, because the Triton-X reacts with Bradford, should I reduce it to 0.05% Triton-X? Would that be enough to lyse mammalian cells?

 

 

 

Yes, 0.05% Triton-X100 can lyse cells very well. Try and check cells under microscope.

 

Critical Micelle Concentration (CMC)for Triton-X-100 is : 0.24mM (0.0155%, w/v). 

0.05% is twice of Critical Micelle Concentration (CMC)for Triton-X-100.

 

http://openwetware.o...entration_(CMC)

 

http://www.piercenet...ergent-solution


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#6 memari

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Posted 09 January 2014 - 12:46 PM

Hi all,

 

However, because the Triton-X reacts with Bradford, should I reduce it to 0.05% Triton-X? Would that be enough to lyse mammalian cells?

 

 

 

If you take 5ul of samples and mix it with 1ml of assay. the concentration of Triton will be 0.005 %.

Triton-X100 at 0.05% concentration interferes with that assay:

 

http://www.bio-rad.c...re/4110065A.pdf


Edited by memari, 09 January 2014 - 12:47 PM.

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#7 Tai

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Posted 17 March 2014 - 11:53 PM

 

Hi all,

 

I am trying to lyse COS-7 cells to detect actin-related proteins in the cytoplasm. My original lysis buffer recipe is:

 

150mM NaCl

1% Triton-X

50mM Tris pH 8.0

Benzamidine HCl 1mM

 

However, because the Triton-X reacts with Bradford, should I reduce it to 0.05% Triton-X? Would that be enough to lyse mammalian cells?

 

 

 

Yes, 0.05% Triton-X100 can lyse cells very well. Try and check cells under microscope.

 

Critical Micelle Concentration (CMC)for Triton-X-100 is : 0.24mM (0.0155%, w/v). 

0.05% is twice of Critical Micelle Concentration (CMC)for Triton-X-100.

 

http://openwetware.o...entration_(CMC)

 

http://www.piercenet...ergent-solution

 

 

 

About CMC, normally we should not have final concentration of components more than this CMC point, right ?  


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