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A homemade device for linear sucrose gradients
Analytical Biochemistry 379 (2008) 211–212
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A homemade device for linear sucrose gradients Daniele Bellavia, Doriana Cellura, Giorgia Sisino, Rainer Barbieri * Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Received 8 April 2008 Available online 17 May 2008
a b s t r a c t We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials—a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained. Ó 2008 Elsevier Inc. All rights reserved.
Linear sucrose gradient centrifugation is a very useful method of fractionating subcellular particles and macromolecules on the basis of their weight and conformation. A linear sucrose gradient is obtained through the use of a relatively simple apparatus (Fig. 1A) consisting of two separate identical cylindrical or conical chambers connected by a tube at the bottom of each chamber. A throttle valve is placed in the connecting tube to prevent contact between the fluids in the chambers before mixing. An outlet tube, also closed by a throttle valve to prevent flow until desired, comes out of the bottom of one of the chambers (chamber 1 in Fig. 1A). A stir bar is placed in chamber 1, which is then placed on a magnetic stirrer plate. Equal volumes of the endpoint sucrose solutions are placed in each chamber, and the connection between the two tanks is opened. Because the heights of the fluids in chambers 1 and 2 are the same, when the valve is opened, there should be no flow from one to the other. However, when the outlet tube is opened, fluid that is to be collected in a centrifuge tube flows out of chamber 1, creating an imbalance in the hydrostatic pressure between the two chambers. As a result of this, fluid from chamber 2 flows into chamber 1 so that the height of the fluids in the two chambers remains the same. If the less dense sucrose solution is placed in chamber 2, this solution will flow into chamber 1. While fluid leaves the system, the solution in chamber 1 is mixed by the stirrer, and this results in a linear gradient of sucrose concentrations, from the most dense to the least dense, leaving the system. For preparation of sucrose gradients, the outlet flow is allowed to enter the bottom of a centrifuge tube through a plastic tube (Fig. 1A). We have developed a simple and inexpensive apparatus to create accurate linear gradients using a phleboclysis tube, a syringe, a magnetic stirrer, a piece of Pongo (Play-Doh), and a flask.
* Corresponding author. Fax: +30 091 6577210. E-mail address: [email protected] (R. Barbieri). 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.05.010
Fig. 1B shows a schematic drawing of our system. A syringe without its plunger (the volume required depends on the volume of the centrifuge tube) is used as a tank for the lower concentration sucrose solution; a common plastic phleboclysis tube with its flow valve connects the syringe to a flask, placed on a magnetic stirrer plate, containing the higher concentration sucrose solution. An outlet piece of phleboclysis tube connects the bottom of the flask with a centrifuge tube in which the gradient is collected, as occurs in the standard apparatus. The system is sealed by bunging the flask with a piece of Pongo (Play-Doh). At the steady state, there are identical levels of sucrose solution in the flask and in the outlet tube. Because the flask system is isolated, when the first drop from the syringe is allowed to fall into the flask, an equal volume of the solution is transferred from the flask to the outlet tube; this process continues until the inlet flux into the flask is stopped. The first drop to fall into the flask solution is mixed by the action of the stirrer and, drop by drop, the flask solution is progressively diluted. This solution is progressively transferred into the tube. The result is a linear sucrose gradient collected in the centrifuge tube, going from the higher sucrose concentration (the one in the flask) collected from the bottom to the lowest sucrose concentration (the one in the syringe) at the top. To demonstrate the accuracy of the gradient we created using our apparatus, we measured the refractive index n of one of every 10 drops collected from a 20 to 50% sucrose gradient using an apparatus for refractometry (New Abbe Refractometer, Atago). The n values obtained were plotted in the graph shown in Fig. 2. The comparison between the n values we measured and the tendency line for endpoint sucrose n values indicates the accuracy of the gradient we obtained. Because most laboratories today are already equipped with a sucrose gradient apparatus, it is difficult to find one in the catalogues of the most well-known biomedical companies. This fact, along with the low cost of our system, makes this method a recommended one for routine laboratory use.
212
Notes & Tips / Anal. Biochem. 379 (2008) 211–212
Fig. 2. Diagram showing the experimental values (triangles), obtained as described in the text, with respect to the tendency line obtained by joining the endpoint sucrose solution values.
Acknowledgment Fig. 1. Schematic drawing of a conventional sucrose gradient apparatus (A) and our device (B).
This work was supported by funds of the Italian Ministero dell’Università e della Ricerca Scientifica.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A homemade device for linear sucrose gradients Daniele Bellavia, Doriana Cellura, Giorgia Sisino, Rainer Barbieri * Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Received 8 April 2008 Available online 17 May 2008
a b s t r a c t We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials—a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained. Ó 2008 Elsevier Inc. All rights reserved.
Linear sucrose gradient centrifugation is a very useful method of fractionating subcellular particles and macromolecules on the basis of their weight and conformation. A linear sucrose gradient is obtained through the use of a relatively simple apparatus (Fig. 1A) consisting of two separate identical cylindrical or conical chambers connected by a tube at the bottom of each chamber. A throttle valve is placed in the connecting tube to prevent contact between the fluids in the chambers before mixing. An outlet tube, also closed by a throttle valve to prevent flow until desired, comes out of the bottom of one of the chambers (chamber 1 in Fig. 1A). A stir bar is placed in chamber 1, which is then placed on a magnetic stirrer plate. Equal volumes of the endpoint sucrose solutions are placed in each chamber, and the connection between the two tanks is opened. Because the heights of the fluids in chambers 1 and 2 are the same, when the valve is opened, there should be no flow from one to the other. However, when the outlet tube is opened, fluid that is to be collected in a centrifuge tube flows out of chamber 1, creating an imbalance in the hydrostatic pressure between the two chambers. As a result of this, fluid from chamber 2 flows into chamber 1 so that the height of the fluids in the two chambers remains the same. If the less dense sucrose solution is placed in chamber 2, this solution will flow into chamber 1. While fluid leaves the system, the solution in chamber 1 is mixed by the stirrer, and this results in a linear gradient of sucrose concentrations, from the most dense to the least dense, leaving the system. For preparation of sucrose gradients, the outlet flow is allowed to enter the bottom of a centrifuge tube through a plastic tube (Fig. 1A). We have developed a simple and inexpensive apparatus to create accurate linear gradients using a phleboclysis tube, a syringe, a magnetic stirrer, a piece of Pongo (Play-Doh), and a flask.
* Corresponding author. Fax: +30 091 6577210. E-mail address: [email protected] (R. Barbieri). 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.05.010
Fig. 1B shows a schematic drawing of our system. A syringe without its plunger (the volume required depends on the volume of the centrifuge tube) is used as a tank for the lower concentration sucrose solution; a common plastic phleboclysis tube with its flow valve connects the syringe to a flask, placed on a magnetic stirrer plate, containing the higher concentration sucrose solution. An outlet piece of phleboclysis tube connects the bottom of the flask with a centrifuge tube in which the gradient is collected, as occurs in the standard apparatus. The system is sealed by bunging the flask with a piece of Pongo (Play-Doh). At the steady state, there are identical levels of sucrose solution in the flask and in the outlet tube. Because the flask system is isolated, when the first drop from the syringe is allowed to fall into the flask, an equal volume of the solution is transferred from the flask to the outlet tube; this process continues until the inlet flux into the flask is stopped. The first drop to fall into the flask solution is mixed by the action of the stirrer and, drop by drop, the flask solution is progressively diluted. This solution is progressively transferred into the tube. The result is a linear sucrose gradient collected in the centrifuge tube, going from the higher sucrose concentration (the one in the flask) collected from the bottom to the lowest sucrose concentration (the one in the syringe) at the top. To demonstrate the accuracy of the gradient we created using our apparatus, we measured the refractive index n of one of every 10 drops collected from a 20 to 50% sucrose gradient using an apparatus for refractometry (New Abbe Refractometer, Atago). The n values obtained were plotted in the graph shown in Fig. 2. The comparison between the n values we measured and the tendency line for endpoint sucrose n values indicates the accuracy of the gradient we obtained. Because most laboratories today are already equipped with a sucrose gradient apparatus, it is difficult to find one in the catalogues of the most well-known biomedical companies. This fact, along with the low cost of our system, makes this method a recommended one for routine laboratory use.
212
Notes & Tips / Anal. Biochem. 379 (2008) 211–212
Fig. 2. Diagram showing the experimental values (triangles), obtained as described in the text, with respect to the tendency line obtained by joining the endpoint sucrose solution values.
Acknowledgment Fig. 1. Schematic drawing of a conventional sucrose gradient apparatus (A) and our device (B).
This work was supported by funds of the Italian Ministero dell’Università e della Ricerca Scientifica.