Mammalian Cell Lysis Buffer Triton-X100 Content - (Jan/07/2014 )

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?

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

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

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.

alphawhiskey on Wed Jan 8 02:42:46 2014 said:

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.org/wiki/Critical_micelle_concentration_%28CMC%29

http://www.piercenet.com/product/triton-x-100-detergent-solution

alphawhiskey on Wed Jan 8 02:42:46 2014 said:

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.com/webroot/web/pdf/lsr/literature/4110065A.pdf

memari on Thu Jan 9 20:40:22 2014 said:

 

alphawhiskey on Wed Jan 8 02:42:46 2014 said:

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.org/wiki/Critical_micelle_concentration_%28CMC%29

 

http://www.piercenet.com/product/triton-x-100-detergent-solution

 

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