• How to use Capillary Counterdiffusion with GCB-Domino
• Frequently Asked Questions

How to use Capillary Counterdiffusion with GCB-Domino

Triana Capillary Counterdiffusion (CCD)™ is a set of reagent kits designed from the basis of the counterdiffusion technique to provide a rational and powerful screening method for the crystallization of biological macromolecules. Triana CCD screening fully exploits the power of counterdiffusion in order to explore the phase diagram, both thermodynamically and kinetically. Triana CCD also has the following advantages:

Triana CCD setting reduces the amount of protein down to 300 nanoliters per experiment.
Triana CCD experimental setting is a straightforward kit that does not need pipetting, grease or drops
Triana CCD is neither a factorial nor a sparse matrix screen. It is a pH versus precipitant screen that always provides information on the crystallisation behaviour of proteins.
Once crystals are obtained, they can be directly diffracted with the Triana CCD capillaries. Without handling, mounting, or doubts nor on the macromolecular nature of the crystals or on their quality.

Kit Composition

Six prefilled GCB-Domino sealed with aluminium tape.
Six black caps.

How to prepare the macromolecular solution

The macromolecular solution should be prepared as pure as possible and used after microfiltration. We recommend the use of a protein concentration of 5 to 10 mg/ml preferably in water or alternatively in a buffered solution at a buffer concentration below 50mM. The sample must be free of any unnecessary additives in order to observe the effect of the precipitants in the kits. Test two protein concentrations per protein. You can also test the effect of divalent metals if you consider it is important for the protein.

How to perform the screening

		Choose the kit you are going to use from the available GCB-Domino screening kits. Prepare the material you will need, i.e, protein solutions, capillaries (0,1 mm Ø) and sealing putty
		Remove the bag containing the black covers and take one GCB
		Cut the aluminium seal as shown in the Figures.
		Dip one capillary into the protein solution. The protein solution will rise by capillarity and the capillaries will be filled. Seal the upper end with the putty.
		Dip the filled capillary into the GCB-Domino. Punch the unsealed end of the capillary across the gel located on top of the precipitant.
		Repeat the procedure with other capillaries filled with protein solution at different concentrations or with certain additives such as divalent metals or detergents. Close the GCB-Domino using the black cap. Repeat the procedure for the remaining five boxes. We recommend you return the six boxes to the cardboard frame considering pH labels.

How to examine the capillaries

The capillaries are examined under a stereo microscope. In order to compare the solubility as a function of pH, we recommend keeping the GCB/domino boxes in the cardboard frame. Examine the capillaries after the first day, after the first week and after the first month. Due to the flat design of the GCB, it can also be used with most of the high magnification optical microscopes with long focal length objectives. If you remove the glass capillaries from the GCB you can observe them under crossed polarisers even at higher magnification. After observation, reinsert capillary into its GCB.
Remember: Counterdiffusion works because it starts far from equilibrium. The result of a CCD experiment evolves along the length of the capillary in time. Therefore, we recommend you record your observation along the tube for a given gross time (days).

References and Readings

J.M. García-Ruiz, Counterdiffusion Methods for Macromolecular Crystallization. Methods in Enzymology, Vol. 368 (2003) 130-154.

Technical Support

Inquiries regarding the use and interpretation of the Triana CCD screen results are very welcome. Please e-mail triana@trianatech.com or fax (+34 958 750 584) your request to Triana Science & Technology. We will get back to you as soon as possible.

Download here the User Guide of the Capillary Counterdiffusion Kits in pdf.

Watch a video showing how to use the Capillary Counterdiffusion Kits.

Frequently Asked Questions

1. How many capillaries can I use per box?
2. Can I test several proteins in the same box?
3. What is the whitish top layer of the precipitant solution?
4. What is the composition of the precipitating solution?
5. Can I transfer the capillary from one box to another?
6. Can I use crystals obtained in the screening for X-ray diffraction?
7. How many times can I use the kit?
8. How long can I use the kits for?

How many capillaries can I use per box?

It depends on your skill. We recommend four or five, but some customers are able to punch up to six per box.

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Can I test several proteins in the same box?

Yes, you can. You can test up to six different proteins with one screening kit. Place each protein solution in a different capillary and punch them in the gel. They will not interfere with each other. Proteins will diffuse slower than the precipitant.

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What is the whitish top layer of the precipitant solution?

It is a gel layer. When the capillary is punched through the gel layer, a short gel plug is formed in the lower part of the capillary. This gel plug ensures that the protein will not leak from the capillary. The gel layer also helps to hold the capillary, providing therefore an easier and better viewing.

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What is the composition of the precipitating solution?

Each kit has a fixed precipitating agent (or combination of precipitating agents) at a given concentration. Each box, belonging to the same kit, is buffered with a different pH value at a concentration of 100 mM. For more details please visit our website.

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Can I transfer the capillary from one box to another?

Yes, you can. For instance, if you have tried a salt kit such as NaCl and the protein solution remains clear, you can transfer the capillary to another kit of a different salt or PEG. When transferring capillaries from one kit to another we recommend you always maintain the same pH value.

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Can I use crystals obtained in the screening for X-ray diffraction?

Yes, you can. First, you must make sure you seal the lower part of the capillary with sealing wax or putty in order to avoid evaporation. Put the capillary directly on the X-ray beam to test if they are protein crystals and verify their quality.

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How many times can I use the kit?

As many times as you wish, for the same or for different proteins, until the solution evaporates or the gel layer loses consistency.

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How long can I use the kits for?

We recommend not to use the kits once the expiry date has passed, usually between two and three months after its manufacture. We are working to improve the duration of the kits.

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