i passed my acetone precipitated protein sample from g-25 sephadex column and downconcentrated to 200 micro liter but i am getting lipid layer on the top while centrifuging it.
however, when i am diluting my sample with miliq water it is getting mixed but again that lipid layer is coming when i am downconcentrating it further.
please give some protocol to remove that lipid layer as i have to pass my protein sample through hplc for purity analysis.
12/21/17
Dear Rahul,
If you are not worried about denaturing your protein, you could add a small amount of hexane to the sample. This will dissolve the lipid. You can then spin the sample at low speed , then very carefully remove the (upper) hexane layer w/ the dissolved lipid. This step can be repeated 1-2 times more to make sure you capture all the lipid. Any residual hexane can be removed by gentle aeration under nitrogen.
If you don't want your protein to come in contact w/ hexane or some other organic solvent, you can dilute the protein solution with DI-water or buffer to a larger volume, e.g., from 0.2 ml to 2.0 mL. Then, insert a very narrow-bore glass pipet, through the lipid layer and carefully draw off the aqueous lower layer with the protein. The protein can then be concentrated using lyophilization. You can easily make a narrow-bore pipet by carefully heating the narrow portion of a Pasteur pipet in a Bunsen burner flame, then drawing it out while it is still soft.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, IA USA [email protected]
The lipids usually end up on top of the liquid layer after being in a refrigerated centrifuge (3,500 - 5,000 x g) with the protein in the pellet. Resuspension with buffer followed by dialysis results in native protein. (When you pour off the supernatent you might need to wipe down the interior of the tube with a Kim wipe to get rid of any residual lipid.)
thanks @W.J. Colonna and @ Entedhar Sarhat ....for your advice and i tried what you all said.
in hexane my protein is losing activity so i cant used that method and even in dialysis i am loosing my lot of sample.
but what i did.... i tried to remove supernatant without disturbing the lipid layer with the help of syringe and tried to accumulate my sample as much as i can but still i am getting very little layer of lipid while keeping in refrigerator.
is there any other way to remove that lipid layer.
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