Thank you for all the replies with various suggestions and guidelines. We will adjust our workflows, and let’s hope the clogging will not appear (at least not so often).
Best regards Cyprian From: CCP4 bulletin board <[email protected]> On Behalf Of Diogo V.M. Melo Sent: Thursday, March 27, 2025 10:46 AM To: [email protected] Subject: Re: [ccp4bb] microfluidizer Nieczęsto otrzymujesz wiadomości e-mail z adresu [email protected]<mailto:[email protected]>. Dowiedz się, dlaczego jest to ważne<https://aka.ms/LearnAboutSenderIdentification> CAUTION: External Sender. Please do not click on links or open attachments from senders you do not trust. I highly recommend you double check the buffer, some ingredients might be dissolved but crash out and clog during the procedure, no matter how viscous or not the sample ends up being. If one of the buffer ingredients behaves as such, you might delay using it or lower the concentration until after the lysis and quickly add it afterwards. Best of luck, Diogo. Rob Wheatley <[email protected]<mailto:[email protected]>> escreveu (quarta, 26/03/2025 à(s) 20:01): 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 <[email protected]><mailto:[email protected]> 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). 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