Hey guys.
I have been working with a hypothetical protein that binds fatty acids (which we don't know what they are) and I can purify this protein without any major problems. Mass spectra as well as other biophysical measurements indicate the presence of ligands that we believe to be hydrophobic (such as fatty acids and lipids).
However, I would like to find protocols to extract these ligands and apply them in TLC. I don't mind disrupting the structure of the protein but I need this extraction to be successful.
Thanks
The process of retrieving lipids from the hydrophobic cavity of a protein typically includes standard procedures such as protein purification, lipid extraction, and structural analysis using methods like X-ray crystallography or cryo-electron microscopy. Protocols may differ based on the unique characteristics of the protein-lipid interaction, and for accurate information, it is advisable to consult relevant literature or seek guidance from experts in the field.
The protein may be precipitated and separated from lipid by adding an equal volume of methanol (equal to the volume of protein solution).
1. Are you wanting to isolate these hydrophobics?
2. Are you starting with crude protein extract or starting with cell lysate? Answering these two questions first might help with the extraction protocol to use next. Here are some GENERAL lipid extraction protocols:
Christelle Anne Fernandez Ancajas My start sample is the recombinant protein that 've been expressing in E.coli.
This protein is co-purified with ligands during the purification process (we saw that through ESI- MS)
We suspect that these ligands are Fatty acids/lipids.
So, answering your question, I want isolate these hydrophobics.
Can you tell from your ESI-MS if you get phospholipid fragmenting into Facyl chains + headgroup + glycerol? or are you getting fragments of just FAcylchains? It's important to consider this because you dont want your lipid extraction to be 'too harsh' depending on your application.
*For simple hydrophobics use Bligh and Dyer method with CHCl3/MeOH
*For phospholipids (if you see evidence of phospholipids as ligands??) use hot ethanol extraction or 4:1 ratio of MeOH/CHCl3 extraction solvent where the extraction was performed twice, followed by a 1:2 ratio of CHCl3/0.1%Acetic acid.
**For acidic phospholipids, use CHCl3/MeOH/12N HCl (2:4:0.1, v/v)
For all of these you end up separating your hydrophobics in one layer and your protein as a pellet which you can recover.
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