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Removal of lipids from cell lysate?

Removal of lipids from cell lysate? - Protocols and Methods Forum

Removal of lipids from cell lysate? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 05-28-2006, 05:19 PM
Alexandra Baer
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Default Removal of lipids from cell lysate?



Dear Kevin,
I found your topic when I searched in google because of the same
trouble.....
However concerning my problem: when I lyse my cells (oligodendrocytes) I too
have lipids in the lysate. THe following western blot detecting higher
molecular weight proteins is not possible because of them....
So I want to ask whether you already found some sollution, because I only
have a kit from calbiochem and I'm afraid that I will lose to much protein,
when I try to get rid of the lipids using it.
So I really hope to find someone who has already experience with this topic.
Thanks very much,

Alex

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Old 05-28-2006, 09:00 PM
DK
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Default Removal of lipids from cell lysate?

In article <[Only registered users see links. ].net >, "Alexandra Baer" <[Only registered users see links. ].at> wrote:

Why?


1. Lipids flotate. Spin the lysate as hard as you can (ultracentrifuge is best
but 20K in JA-20, 1 hr will work OK too). Remove flotating lipid with Q-tip,
etc.

2. Add an extra SDS (2X over normal) to the SDS-PAGE loading buffer.
Problem with excess lipids and detergents is that they compete with SDS,
giving not as nice gels.

OR

3. Do an chloroform/ethanol precipitation of your sample(s). This will remove
lipids and detergents from your sample nearly quantitatively.


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