Hi everyone. Just out of curiosity. I am using High Five insect cell as the protein expression system. Cells are resuspended in 50mM Tris-HCl (pH 8.4), 200mM NaCl, 5% glycerol. After cell sonication and then centrifugation (20,000rpm, Beckman JA-20 rotor), there is always some white floating stuff on the surface of supernatant (in the pictures attached). Anyone has any idea about what this might be? Thanks!
I believe it to be lipids. If you want to remove it, avoid disturbing the layer when you move the centrifuge tubes so that it remains concentrated, then use a very small transfer pipette with a narrow tip to gently draw it off the surface. If it does not cause any problems for subsequent steps, then you can probably just ignore it.
I believe it to be lipids. If you want to remove it, avoid disturbing the layer when you move the centrifuge tubes so that it remains concentrated, then use a very small transfer pipette with a narrow tip to gently draw it off the surface. If it does not cause any problems for subsequent steps, then you can probably just ignore it.
Adam B Shapiro is correct, to my knowledge. This is an example of oil and water do not mix, or hydrophobic compounds don't dissolve in water. Any lipids, such as those from membranes, including from organelles, will collect at the surface of the aqueous layer. Other classes of lipids will also be found in this layer, which is less dense than water. Membrane-bound proteins will also likely be found in this layer, though depending on their properties, they may at the interface between this layer and water or all the way inside the top layer. The lipids and lipoproteins can be extracted with organic solvents, though I would follow a published protocol for the best results- there are many lipidomics and related papers these days. For example: Chapter Basic Techniques for Lipid Extraction from Tissues and Cells
Dear Colleagues Jian Wang Adam B Shapiro James Keane Bashkin
Thanks for the interesting question
Hydrophilic lipids should be separated with proteins into fractions in a centrifuge. Hydrophobic to separate easier. In general, depending on what your task is. If you know that you need not to spoil the fraction of proteins or lipids, you can choose the appropriate solvents. Regards, Pushkin Sergey Viktorovich
- Badges
- Science topic
- Similar topics
- Biological Science
- Zoology
- Invertebrate Zoology
- Arthropods
- Insects
- Insect