Overall when using the Microfluidizer, while clogging is definitely
annoying, I have never found it a major problem. My metric is: the time
I spend unclogging the machine is less than the time an extra step to
prevent clogging would add to the protocol. :) I am guessing on average
I have processed multiple liters between clogs.
What interaction chamber are you using? For cell lysis, (E. coli,
yeast, etc.) my testing has always found an H10Z (100 um) more than
sufficient. Note that chambers differ in both channel size and
geometry (Y or Z configuration). Speaking of chambers, with the LM20
you can also add an APM (auxilliary processing module) upstream of the Z
interaction chamber to preprocess the sample. In my experience the
Microfluidics sales reps have always been quite helpful in letting you
test various chambers without purchasing these rather costly items.
Besides the actual equipment, I would think about the preceding steps.
If the cells are harvested by centrifugation, perhaps preparing a more
loosely packed pellet (shorter centrifugation time, lower rcf) would
make it easier to resuspend? In general with a relatively stable
proteins I do stir the suspension on ice for a while, until most clumps
are disrupted. One other thing that might help with frozen E. coli
cells is adding some nuclease while resuspending the cells to break up
the 'gloopiness' formed by DNA release during the freeze/thaw cycle.
Good luck,
Rob
On 3/25/2025 12:43 AM, M T wrote:
Dear Cyprian,
I recommend to users to use a Potter-Elvehjem homogenizer before to
use the LM20, or even (in case of non fragile protein) to do some
cycle of sonicator.
I advice also to pay attention to the anti-drop ring of the used
bottle. Because in case of old one, you may have some plastic
particles which falls in the sample and clog the LM20 cell. Of course,
dust is not welcome neither…
To unclog the cell there is no other choice than to invert it. In case
of severe clogging, the water-bath sonicator can help (gentle heating
and sonication), before to retry to unclog the cell mounted in a
reverse way.
Best.
Michel.
Le 25 mars 2025 à 07:09, Cyprian Cukier <cyprian.cuk...@selvita.com>
a écrit :
Dear Community,
First of all, apologies for the slightly off-topic question, which is
related to protein purification rather than crystallography.
I am seeking your advice on preparing the cell pellets for the lysis
process. We have an LM20 microfluidizer and we experience quite
frequent clogging of the system that is tedious to remove. This
happens particularly with the E. coli pellets, but from time to time
the eukaryotic cells are also problematic. Does anyone have
well-established protocols on how to handle the cell pellets before
applying them to the lysis process in the microfluidizer?
I should add that of course we follow the manufacturer's
recommendations to have a homogenous suspension, which is not too dense.
I would also appreciate any advice on how you clean your systems
(regularly or in case of clogging).
Best regards
Cyprian
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