Crystallization robot and trypsin

From: Horacio Botti
Date: 24 January 2012 12:12

Dear all

We may use a Honeybee 963 robot to screen crystallization conditions for trypsin-containing protein samples and we are worried about robot contamination by residual protease.
How do you normally clean robots when using this kind of sample? Your suggestions/recommendations will be appreciated. Thks!!

Horacio Botti
Unit of Protein Crystallography,
Institut Pasteur of Montevideo, Uruguay.


PS: below you have an old short CCP4 discussion:

[ccp4bb]: Crystallization robots and protease.

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• From: Marc Graille
• Date: Mon, 19 Dec 2005 13:21:36 +0100

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Dear all,

I have a question regarding the use of robotics to screen for crystallization conditions for proteases.
Does anyone have already used robots on proteases? If yes, have you experienced any protease "contaminant" in the robot pipes, which could have affected the results on other projects performed during the next few days ??
I mean that we cannot exclude that a "contaminant" could digest the protein we are working on and yield crystals of a fragment of the studied protein.

We are hesitating in using our crystallization robots on proteases as we are afraid to have some contaminant in the pipes that will disturb all our future experiments!!!
Any advice about how to clean the robot syringes after use of proteases are welcome!!!


Regards,

Marc

-- Marc Graille, PhD

  Dear Marc  We recommend cleaning dispensing tips with Hellmanex II from the German company Hellma.  It's for cleaning cuvettes.  You can buy it from VWR and others.  Hellmanex is a mildly alkaline solution with surfactants etc.  It has no enzymes in it, but you do need to flush with buffer to get rid of the alkali.  Previously, users reported that cleaning with methanol mixed with concentrated HCl worked very well in extreme cases.  Our robot, the Oryx, uses only one tip for protein.  This is semi-disposable.  Using one tip and touching off the drops has the great advantage that virtually no protein is wasted, and drops as low as 20 nl can be dispensed, even containing 50% glycerol!  We recommend that users keep tips that have been used for proteases separately, and clean them after use.  (The system comes with 8 tips and replacements cost 45 USD.)  Also we recommend that tips are thoroughly cleaned once a fortnight for average use in any case.  I hope this is helpful.  Sincerely  Patrick 

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From: Regina Kettering

We have a Honeybee system but do not usually use proteases.  The biggest problem we have found is that if anything precipitates in the tips they have to be washed very well, usually with water or ethanol.  The ceramic tip can be washed using low concentrations of HCl (0.1M), which I believe would also alleviate the protease problem.

Regina

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From: Sebastiano Pasqualato

Hi Horacio,

we have a Cartesian Honeybee and perform trypsin-containing trials by setting up drops with a dedicated protein+trypsin tip, which we then wash extensively with water, 6 M guanidinium hydrochloride, then water, isopropanol and again water.

hth,

ciao,

Sebastiano

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From: Bart Hazes

At some point we aspirated an expression plasmid, let it sit in the tip for a while and then went through different washing protocols, water, high-salt, 0.1M NaOH, and then dispense a small volume of water into a PCR reaction. We found just a water wash got rid of most of the DNA, as long as the outside of the tip was also washed. After the high salt we could no longer detect the DNA by PCR. Although protein is not the same as DNA I would have no concern about protease contamination if you implement a good wash/strip protocol for the protein dispense tip.

Bart