Sucrose in Lysis Buffer? - (Jan/22/2007 )
Hello.I want to ask one question about lysis buffer.
What is the function of sucrose in lysis buffer ?
Is it necessary to add sucrose when making lysis buffer?
If i remember well, sucrose is to prevent the lysis of lysosome.
I don't use it, however I use protease inhibitors.
What is the function of sucrose in lysis buffer ?
Is it necessary to add sucrose when making lysis buffer?
increases osmolarity; stabilzes integrity of membranes
sucrose is used in buffers for subcellular fractionation.
Sucrose is used in buffers for subcellular fractionation...
Can I make the lysis buffer as follows?
50mM Tris-HCl, 150mM NaCl, 5mM EDTA, 1% Triton-X 100, AND ....% Sucrose.
Then What about the percentage of the sucrose?
Sucrose is used in buffers for subcellular fractionation...
Can I make the lysis buffer as follows?
50mM Tris-HCl, 150mM NaCl, 5mM EDTA, 1% Triton-X 100, AND ....% Sucrose.
Then What about the percentage of the sucrose?
if you like to do subcellular fractionation, then 1% triton x-100 will lyse also your organelles and is unsuitable for subcellular preparations but we do not know what you really like to perform...
It have 2 purpose 1st the one that say the bearer and 2nd because it increase the viscosity and so when centrifuge it will mantein the nucleic acids in the supernatant when discarting the cell debris.
we use 25mM tris-cl ( pH 8.0) and 0.25M sucrose and no triton for subcellular fractionation.
In general the lysis buffer will contain a detergent like triton to solubilize the membranes, sucrose for the reasons above, enzymes to eliminate something that you don't want, for example is you want DNA only then add Rnase and protease, if want protein only then add a nuclease and protease inhibitors to protect proteins and so on.
Thank you for your kind replies.
I want to perform subcellular frationation and analyze the kinase activity.