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WO2005026715A1 - Focalisation isoelectrique serielle de gradients de ph immobilises - Google Patents

Focalisation isoelectrique serielle de gradients de ph immobilises Download PDF

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Publication number
WO2005026715A1
WO2005026715A1 PCT/EP2004/010090 EP2004010090W WO2005026715A1 WO 2005026715 A1 WO2005026715 A1 WO 2005026715A1 EP 2004010090 W EP2004010090 W EP 2004010090W WO 2005026715 A1 WO2005026715 A1 WO 2005026715A1
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WO
WIPO (PCT)
Prior art keywords
gradients
immobilized
gradient
ief
voltage
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Ceased
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PCT/EP2004/010090
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German (de)
English (en)
Inventor
Helmut Zengerling
Gerhard Schwall
Michael Cahill
Slobodan Poznanovic
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ProteoSys AG
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ProteoSys AG
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Publication of WO2005026715A1 publication Critical patent/WO2005026715A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the invention relates to immobilized pH gradients, devices containing them and a method for producing serial immobilized pH gradients, in particular for isoelectric focusing.
  • Zone electrophoresis b) Isotachophoresis c) Isoelectric focusing
  • Zone electrophoresis involves glycine, bicine and tricine buffered sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for proteins, which is routinely used for the study of proteins (Wilkins et al. 1998).
  • Isotachophoresis uses hydrophilic matrix materials and typically shows high resolution but low loading capacity. In combination with free-flow electrophoresis, this method can be used for micro-preparative purposes (Weber and Bocek, 1998). Isoelectric focusing (IEF) is carried out either in liquid density gradients, in gel gradients (Righetti, 1990) or in multi-chamber devices with immobiline-based separating gel media (Righetti et al., 1997).
  • the first developed method uses a pre-focusing of carrier ampholytes (TA) to their isoelectric point and a subsequent focusing of proteins.
  • TA carrier ampholytes
  • the proteins separated in this way are then separated in a second dimension in an SDS-PAGE (Klose, 1975; Klose and Kobalz, 1995; O'Farrel, 1975).
  • This system has an excellent resolution and, when using 46 cm TA-IEF gels, achieves a separation of over 10,000 protein spots per 2D-PAGE gel (Klose and Kobalz, 1995).
  • a later developed system for isoelectric focusing uses acrylamide-like acrylamido buffer monomers (immobilins) that carry a functional buffer group.
  • immobilins acrylamide-like acrylamido buffer monomers
  • a pH gradient is created by formed the formation of a concentration gradient of gel solutions with different pH values. After the gradient has been formed, the acrylamido buffer monomers polymerize together with the aryl amide matrix and are thus immobilized (Görg et al., 2000; Görg et al. 1988; Righetti, 1990).
  • IPG immobilized pH gradients
  • These immobilized pH gradients (IPG) gels are poured by building up a density gradient with glycerol between the two solutions of different pH values. Since this process is imitated by diffusion, the real estate gradients produced are typically less than 30 cm long to ensure a correct density gradient. Therefore, no immobilized pH gradients longer than 30 cm have been reported. The longest commercially available IPGs are 24 cm long and
  • the aim of the invention is therefore to provide immobilized pH gradients, devices containing them and a method for producing immobilized pH gradients which at least partially solve the disadvantages existing in the prior art.
  • immobilized pH gradients can be serially connected to one another via an electrically conductive connecting element / connecting bridge, and that the immobilized pH gradients connected in this way have excellent properties for isoelectric focusing.
  • the invention thus relates to immobilized pH gradients, in particular for isoelectric focusing, at least two, preferably a large number, of the pH gradients each being connected to one another in series with at least one electrically conductive connecting element.
  • the connecting element does not adversely affect the immobilized pH gradients, i. H. z. B. restricts the movement of analytes, carrier ampholytes and components of the experimental system between the various immobilized pH gradients.
  • the connection elements can be made of any material, connection and / or mixture, etc., which meet these requirements.
  • these can be, inter alia, polyacrylamide, agarose and / or buffer-containing connecting elements, immobilized pH gradients and / or a filter paper strip which connect the pH gradients to one another so that the free movement between the various immobilized pH gradients is ensured.
  • immobilized pH gradients are furthermore claimed, in which the pH gradients have different pH ranges.
  • These pH ranges can have any pH from pH 0 to pH 14. They advantageously have a “physiological” pH range, ie the range from pH 2 to pH 12, in particular pH 4 to pH 9, on what in particular when analyzing naturally occurring analytes such as.
  • B. Protects a sufficiently good separation and is therefore particularly well suited for isoelectric focusing. If you work in this pH range, you can, for example, create different pH gradients in which there are mixtures from pH 2 to pH 12. So z. B.
  • an immobilized pH gradient the pH range from pH 2 to pH 5, the next the pH range from pH 5 to pH 7, the next pH 7 to pH 9 and the last immobilized pH gradient the range from pH 9 cover up to pH 12.
  • the individual immobilized pH gradients advantageously have no overlapping pH ranges. This can always be advantageous if analytes are to be separated that have very similar pH values and are therefore very difficult to separate from one another.
  • immobilized pH gradients with a (single) length of at least 15 cm, preferably at least 30 cm, in particular at least 50 cm, are also used.
  • Each individual pH gradient should have the specified minimum length.
  • the claimed pH gradients together preferably have at least a total length of 20 cm, preferably at least 60 cm, in particular at least 1 m.
  • the length of the individual pH gradients can then vary, and different lengths of the individual pH gradients are also possible, provided that the sum of the individual pH gradients has the desired total length.
  • the immobilized pH gradients according to the invention can in principle have any length, regardless of the individual and / or total length.
  • IPGs Long to extremely long IPGs can be made with the present invention. It is a characteristic property of these IPGs that these immobilized pH gradients are at least> 1 cm to preferably several meters long, lengths from approximately 15 cm being preferred. As already explained above, each individual pH gradient can have these lengths, or the sum of all pH gradients can have these lengths. It is difficult to create such long individual gradients. In order to create a long individual gradient, a slow, pulsation-free flow rate of the individual, at least two, buffer components must be ensured during the casting process. Again regarding the special conditions for the production long immobilized pH gradient on the scientific publication by Tru et al. directed.
  • a device for isoelectric focusing (IEF) with a so-called IEF chamber and a power supply for providing a focusing voltage for at least one immobilized pH gradient is also claimed, the device having at least two of the described immobilized pH gradients.
  • the IEF chamber is the chamber in which the isoelectric focusing is carried out.
  • the power supply is preferably arranged completely in the IEF chamber.
  • the IPGs can also be surrounded by an electrically insulating material that can be solid or liquid.
  • the device can thus be operated alone or in combination with the power supplier, one or more cables and / or further components for isoelectric focusing.
  • This device alone as well as all conceivable combinations of this device with one or more further component (s) for the isoelectric focusing are hereby disclosed and made the subject of the present invention.
  • the device can have a power supply that delivers a maximum focusing voltage of at least 10 kV, preferably at least 20 kV, in particular at least 35 kV.
  • a power supply that delivers a maximum focusing voltage of at least 10 kV, preferably at least 20 kV, in particular at least 35 kV.
  • This enables the use of long, serially connected pH gradients, in particular with the lengths described above, which greatly improves the resolution that can be achieved.
  • the invention also encompasses any power supply, regardless of the voltage supplied. If desired, a device that supplies lower voltages, ie voltages ⁇ 10 kV, or a device that supplies higher voltages, ie voltages> 35 kV, can therefore also be used as the power supplier.
  • the power supply can be coupled to the IEF chamber via at least one high-voltage-resistant cable which has a dielectric strength that is adapted to the maximum focusing voltage.
  • the power cables between the power supply and the IEF chamber can preferably be permanently installed during the IEF, ie they cannot be removed.
  • the electricity supplier is fully integrated in the IEF apparatus and is provided with a mechanism which switches the electricity supplier off as soon as the chamber is opened and thus ensures that no user of the apparatus is endangered by the current applied to the apparatus.
  • the apparatus can preferably have a locking means which prevents the IEF chamber from opening for a limited time, generally at least 2 minutes, after the power supply has been switched off, until a remaining high voltage has reduced to a non-hazardous value.
  • the device according to the invention is to be operated under high voltage, it is advantageous if cables are used which are suitable for the high voltage to be applied, in particular voltage-isolating, so that no dangerous voltage flashovers occur during operation.
  • the electrically conductive part of the cable can be completely isolated from the ambient air.
  • the voltage to be applied depends crucially on the desired focusing time. The higher the voltage, the faster the analytes to be analyzed, e.g. B. proteins are separated.
  • heat can develop, e.g. B. in the analysis of anal lytes such as B. proteins can possibly lead to carbamylation by the urea in the buffer. Therefore, if a high voltage is not necessary for reasons of time, the device according to the invention can also be operated with low voltage, ie voltages from approximately 150 V to approximately ⁇ 10,000 V.
  • a method for producing immobilized pH gradients in particular for isoelectric focusing, is claimed according to the invention, wherein at least two, preferably a plurality, of the pH gradients are in each case connected to one another in series with at least one electrically conductive connecting element.
  • the connecting element enables the current to flow between the immobilized pH gradients.
  • the connecting element is advantageously a polyacrylamide, agarose and / or buffer-containing connecting element, an immobilized pH gradient and / or a filter paper strip. These components ensure the free flow between the immobilized pH gradients.
  • Other materials, compounds, mixtures, etc. for the connecting element according to the invention are known to the person skilled in the art and are also claimed here.
  • the process according to the invention is further characterized in that the pH gradients have different pH ranges, preferably pH 2 to pH 12, in particular pH 4 to pH 9.
  • any pH range can be used for the system according to the invention, but the “physiological” pH range from pH 2 to pH 12 is distinguished by the fact that it is used in the separation of naturally occurring analytes, such as, for example, Proteins, is particularly suitable.
  • the method according to the invention is further characterized in that the pH gradients each have any desired length of> 1 cm, but preferably each have at least a length of 15 cm, preferably at least 30 cm, in particular at least 50 cm.
  • the pH gradients can furthermore have at least a total length of 20 cm, preferably at least 60 cm, in particular at least 1 m.
  • the length of each individual pH gradient can vary and can be of any conceivable length.
  • the advantage of this system is obvious.
  • commercially available pH gradients, but also (in the laboratory) self-made pH gradients can be freely combined with one another without too much being based on predetermined minimum standards such as For example, the length of the individual pH gradients and their combination options must be taken into account. This considerably reduces the preparative and financial outlay for producing the pH gradients with the desired length.
  • the present invention can also be used to perform isoelectric focusing (IEF) of analytes, such as. B. proteins in an extremely long immobilized pH gradient, the copolymerization of a preformed pH gradient in a matrix such. B. polyacrylamide or a polymer matrix with similar polymerization chemistry of monomers (similar to acrylamide and bisacrylamide).
  • IEF isoelectric focusing
  • the isoelectric focusing is therefore carried out under low voltages of at least 150 V, preferably at least 200 V, in particular 600 V. With these comparatively low voltages, the generation of discharge sparks as well as a possible one Heat development can be counteracted, which would interfere with the isoelectric focusing process. Furthermore, these potential tensions can reduce the potential danger for employees. If you work with low voltages, comparatively long focusing times must be expected.
  • the isoelectric focusing can preferably also be carried out under high voltages of at least 3,000 V, preferably at least 10,000 V, in particular> 20,000 V.
  • a focusing time of approx. 20 h can be expected when applying 10,000 V; with a voltage of 20,000 V this focusing time is reduced to approx. 10 h, with a voltage of 30,000 V to approx. 7 h. Therefore, the use of high voltages during the IEF of long IPGs represents an inventive achievement which greatly increases the throughput and furthermore reduces the spot size of the proteins focused at their isoelectric point (pl).
  • the high voltages are made possible in particular by the use of an electrically insulating medium, in particular an oil, that surrounds the electrodes and the IPGs and / or IPG strips during the IEF.
  • a suitable substance is the dimethylpolysiloxane oil AK10 from Wacker-Chemie GmbH, 84489 Burghausen, Germany, which has insulation properties of better than 10 14 ohm cm.
  • the immobilized pH gradients are focused under an oil or similar water-impermeable layer, since the immobilized pH gradients would otherwise dry out slowly over time due to water loss to the atmosphere.
  • the resolution capacity of the current state of the art IPGs and / or IPG strips and the resulting 2D-PAGE gels limit the number of proteins that can be detected. Because of this low resolution, only a similar number of proteins can be detected with less sensitive detection methods (eg silver staining or fluorescence staining) or with highly sensitive detection methods (eg radio imaging).
  • the molecules to be investigated are marked, separated by 2D-PAGE and detected with a suitable detector.
  • a suitable detector two or more samples with different radio-isotopes or different mixtures of radio-isotopes, or different stable isotopes or fluorescent dyes or mass-different reagents are marked and separated together in a 2D-PAGE gel and then separately with suitable detectors are detected.
  • a detector is required which can distinguish between the differently labeled samples on the basis of physical properties and / or half-lives of the labeling reagents used.
  • the immobilized pH gradients can be cut into so-called IPG strips (immobilized pH gradient strips) of any width after casting. These can then be stored until use, preferably at -20 ° C, or used directly for isoelectric focusing.
  • the sample to be analyzed, in particular proteins is then preferably introduced into the IPG strips by means of active rehydration, ie. H. loaded. In this process, the proteins become active during the rehydration of the dry IPG strips in a rehydration chamber, at low voltages, typically less than 1250 V.
  • the IPG strip is then often transferred to a second chamber which contains electrodes that are spaced relatively far apart.
  • the contact to the electrodes is made via conductive elements (media) (eg filter paper strips soaked with aqueous, chemical-containing solutions) at both ends of the IPG strips.
  • the filter paper strips are replaced at least once during the IEF in order to remove ions accumulating in the filter paper. The removal of these ions accumulating in the filter paper is advantageous for high-quality results.
  • the pictures show:
  • 2A-2C Series of IEF experiments with pig liver proteins (alkylated on cysteine residue with iodoacetamide).
  • Pig liver proteins were extracted and prepared for the IEF according to Vuong et. al (2000) with the modification that the proteins were not iodized.
  • 150 ⁇ g protein are used to rehydrate the IPG strips.
  • the rehydration was carried out in the pH 4-5 IPG using the passive rehydration method.
  • the strips were then transferred to a second chamber.
  • the contact between the ends of the IPG strips and the electrodes was determined using 7 M urea, 2 M thiourea, 4% chaps (3 - ((3-cholamidopropyl) dimethylammonium) -1-propanesulfonate), 1% Triton X-100 and 65 mM dithiothreitol (DTT) impregnated filter paper strips.
  • the following program was used to focus the samples:
  • FIG. 1 shows a schematic representation of a serial IPG-IEF, as was also used for the experiments with pig liver proteins (FIGS. 2A-2C).
  • (1) represents the anode and (2) the cathode of the system.
  • the optional filter paper strips (electrode paper) soaked with buffer are represented by (3) and (4).
  • A, B and C stand for the individual immobilized pH gradients.
  • A is an immobilized pH gradient pH 4-5
  • B is an immobilized pH gradient pH 5-6
  • C is an immobilized pH gradient pH 6-9.
  • AB and BC are the connecting elements for the individual IPGs.
  • 2A-2C show 2D gels of 150 ⁇ g protein extra kt from pork liver focused on a serial IEF gel pH 4 to pH 9 alkylated on the cysteine residue with iodoacetamide.
  • Commercially available 18 cm IPGs from Amer- sham Biosciences, Freiburg, used (pH 4-5, pH 5-6, pH 6-9). Each commercial IPG is shipped with a manufacturer's recommended carrier ampholyte buffer. In the example, each IPG was rehydrated in its recommended buffer. However, the same results are obtained when the buffers are mixed together and each IPG in the buffer mixture is rehydrated.
  • the connecting elements AB and BC consist of 4% polyacrylamide monomers (without acrylamido buffer).
  • the dried polyacrylamide gels were cut into the appropriate shape and rehydrated in IEF buffer.
  • Other possible materials for the connecting elements are e.g. B. buffer-soaked agarose, buffer-soaked paper strips or buffers without a supporting matrix (ie without diacrylamide matrix).
  • the connecting element should preferably not negatively influence the IEF and should ensure the free movement of proteins, carrier ampholytes and components of the experimental system between the IPGs.
  • the pig liver proteins were loaded by rehydration on the acidic IPG (pH 4 to 5) and the isoelectric focusing was then carried out.
  • the voltage was limited to 50 ⁇ A per gel.
  • the temperature was kept at 20 ° C and CO 2 was excluded by immersing the IPGs under oil, coating the oil with argon and trapping the CO 2 with filter paper strips which had previously been soaked in 2 M NaOH.
  • Identical results were obtained when the pig liver proteins were loaded at the basic end of the IPGs (pH 6 to 9), which clearly shows that the protein transfer between the connecting elements is efficient.
  • the examples show commercially available IPGs, but a variety of combinations of high and low resolution IPGs are possible.
  • Electrophoresis gel media the State of the art. J. Chromatog. B 699, 63-75. Vuong GL, Weiss SM, Kammer W, Priemer M, Vingron M, Nordheim A, Cahill MA. (2000). Improved sensitivity proteomics by postharvest alkylation and radioactive labeling of proteins. Electrophoresis. 21, 2594-605.

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Abstract

L'invention concerne des gradients de pH immobilisés mutuellement reliés de façon sérielle, des dispositifs contenant ces gradients, ainsi qu'un procédé de production de gradients de pH sériels immobilisés, en particulier pour la focalisation isoélectrique.
PCT/EP2004/010090 2003-09-12 2004-09-10 Focalisation isoelectrique serielle de gradients de ph immobilises Ceased WO2005026715A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10343334.1 2003-09-12
DE2003143334 DE10343334A1 (de) 2003-09-12 2003-09-12 Serielle isoelektrische Fokussierung von immobilisierten pH-Gradienten

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WO2005026715A1 true WO2005026715A1 (fr) 2005-03-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007073293A1 (fr) * 2005-12-22 2007-06-28 Ge Healthcare Bio-Sciences Ab Gel pour mise au point isoelectrique
CN101294930B (zh) * 2007-04-27 2013-08-14 杨春 一种整体型固定化pH梯度的制备方法及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1907833B1 (fr) * 2005-07-05 2010-01-27 Partnership Bioactivity Separation d'analytes par masse moleculaire et charge
US20080272002A1 (en) * 2007-05-03 2008-11-06 Protein Forest, Inc. System and Method for Proteomics

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068225A1 (fr) * 2000-03-15 2001-09-20 Proteosys Ag Focalisation isoelectrique pour micropreparation
WO2003046513A2 (fr) * 2001-11-27 2003-06-05 Royce Technologies Llc Procede d'electrophorese bidimensionnelle sur gel pour presentation de proteines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068225A1 (fr) * 2000-03-15 2001-09-20 Proteosys Ag Focalisation isoelectrique pour micropreparation
WO2003046513A2 (fr) * 2001-11-27 2003-06-05 Royce Technologies Llc Procede d'electrophorese bidimensionnelle sur gel pour presentation de proteines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GÖRG A ET AL: "The current state of two-dimensional electrophoresis with immobilized pH gradients.", ELECTROPHORESIS, vol. 21, no. 6, April 2000 (2000-04-01), WEINHEIM, pages 1037 - 1053, XP002308489 *
POLAND J. ET AL: "Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.", ELECTROPHORESIS, vol. 24, no. 7-8, April 2003 (2003-04-01), WEINHEIM, pages 1271 - 1275, XP002308713 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007073293A1 (fr) * 2005-12-22 2007-06-28 Ge Healthcare Bio-Sciences Ab Gel pour mise au point isoelectrique
US8858771B2 (en) 2005-12-22 2014-10-14 Ge Healthcare Bio-Sciences Ab Gel for isoelectric focusing
CN101294930B (zh) * 2007-04-27 2013-08-14 杨春 一种整体型固定化pH梯度的制备方法及其应用

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