We are using a Sigma kit for free and total cholesterol determination. First step is homogenization in isopropanol:chloroform:NP40 buffer to extract lipid. The tissue congeals when buffer is added and won't disperse. Prehomogenizing in a small bit of PBS breaks the liver up, but it still congeals when buffer is added. Any suggestions appreciated.
I'd suggest you homogenize with isopropanol first, then add the chloroform and NP40 and homogenize again.
Organic solvents will precipitate great many proteins, this cannot be avoided irrespective of what order you use. If you are only concerned with lipid extraction, the ancient method of using high speed blender (with sharp blades) works quite nicely.
Think of making smoothies! :)
Just be sure to use a good blender with which designed to handle solvents so sparks from motor do not ignite fumes or spills.
Thanks Tausif. We were not planning on grinding that much liver, but we will if if nothing else works :-)
Susan,
Waring blender has a smaller stainless 75 Gram Pulverizer that will work quite well for smaller amounts of tissue. You can easily use it with a few grams of liver along with solvents. I have used it many times a few decades ago.
Homogenization with Polytron works perfectly using 100 ug liver aliquots and PBS (1:1 to 1:10 w/v), and then a standard extraction of lipids following the Bligh&Dyer method or exraction with isopropanol (1:4, v/v).
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