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WO2019121263A1 - Procédé pour séparer différents peptides bêta-amyloïdes par électrophorèse sur gel - Google Patents

Procédé pour séparer différents peptides bêta-amyloïdes par électrophorèse sur gel Download PDF

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Publication number
WO2019121263A1
WO2019121263A1 PCT/EP2018/084629 EP2018084629W WO2019121263A1 WO 2019121263 A1 WO2019121263 A1 WO 2019121263A1 EP 2018084629 W EP2018084629 W EP 2018084629W WO 2019121263 A1 WO2019121263 A1 WO 2019121263A1
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WO
WIPO (PCT)
Prior art keywords
gel
buffer
kit
sarcosyl
gel electrophoresis
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2018/084629
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German (de)
English (en)
Inventor
Jens Wiltfang
Hans-Wolfgang Klafki
Petra Rieper
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaetsmedizin Goettingen Georg August Universitaet
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Universitaetsmedizin Goettingen Georg August Universitaet
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Publication of WO2019121263A1 publication Critical patent/WO2019121263A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis

Definitions

  • the invention relates to a method for the separation of carboxy-terminal variants of Abeta peptides by gel electrophoresis. In particular, it is about the separation of Abeta peptides, d. H. Amyloid-beta peptides, A ⁇ 1-37, A ⁇ 1-38, A ⁇ 1-39, A ⁇ 1-40 and A ⁇ 1-42, which, in particular in terms of their relative concentrations, are potential biomarkers for various neurodegenerative diseases.
  • the stoichiometric loading of the Abeta peptides with SDS (SDS / Abeta molar ratio) in the presence of 8 M urea is proportional to the length of the carboxy-terminal, hydrophobic region of the Abeta peptides. This explains why carboxy-terminal longer variants the Abeta peptides show higher electrophoretic mobility, ie cover in SDS-PAGE in the same time more stretches in the separation gel than shorter variants of Abeta peptides.
  • the gel electrophoresis is carried out in the presence of sodium lauroyl sarcosine (sarcosyl).
  • MIRCERA methoxy polyethylene glycol epoetinum beta
  • SAR PAGE sarcosyl-polyacrylamide gel electrophoresis
  • the sarcosyl can be introduced into a collection gel and / or into a separating gel and / or into a cathode buffer and / or into an anode buffer of the gel electrophoresis.
  • the sarcosyl is incorporated into both the stacking gel and the separating gel as well as at least the cathode buffer.
  • the introduction of the sarcosyl into the collection gel and the separating gel in the usual way may mean that the sarcosyl is already present in the gel when polymerizing the respective gel, i. H. is polymerized into the respective gel.
  • the concentration in which the sarcosyl is present in the method according to the invention in gel electrophoresis typically falls within a range of 0.1% and 0.8% (w / v). A concentration of about 0.25% (w / v) has been found to be particularly practical. At this concentration, the sarcosyl can be introduced into the collection gel and / or into the separation gel and / or into the cathodic buffer and / or into the anode buffer of the gel electrophoresis.
  • the addition "(w / v)" in the concentration of sarcosyl indicates that the percentage concentration indication corresponds to the weight of the sarcosyl in grams relative to the volume of each gel or buffer in 100 ml.
  • concentrations with the addition are given in percent by volume or in ml of the respective substance in 100 ml of the respective gel or buffer.
  • concentrations are given in percent by volume or in ml of the respective substance in 100 ml of the respective gel or buffer.
  • the object of the invention to manage without urea, in particular without urea in higher concentrations, is completely solved by the method according to the invention.
  • the collection gel, the separation gel, the cathode buffer, the anode buffer and also a sample buffer of the gel electrophoresis may be urea-free.
  • urea-free means that there are no functional amounts of urea.
  • These gels or buffers need not be pure urea, i. H. also free of urea traces.
  • gel electrophoresis is carried out in the presence of sarcosyl, and sarcosyl is believed to stoichiometrically load the Abeta peptides as a function of the length of their respective carboxy-terminal region, thus leading to their differential electrophoretic mobility in gel electrophoresis .
  • sarcosyl is a detergent, it serves at least not primarily for the digestion of the respective sample.
  • a sample buffer for gel electrophoresis which is sarcosyl-free and is introduced into the SDS or LDS as a disintegrating detergent.
  • the collection gel and the separating gel as well as the cathode buffer and the anode buffer of gel electrophoresis can be free of SDS and LDS in the method according to the invention, which is also preferred.
  • the SDS and / or LDS in a typical concentration between 0.5% and 2% (w / v), d. H. of about 1% (w / v).
  • sucrose in a concentration between 5% and 20% (v / v) and / or the sucrose in a concentration between 5% and 20 % (w / v) is introduced into the sample buffer.
  • sucrose tends to be favored over that of glycerol because it leads to sharper bands in which the individual carboxyterminal variants of the Abeta peptides are shown after gel electrophoresis.
  • a beneficial factor in the practical testing of the method according to the invention has continued to be a jump in the pH between the collection gel and the separating gel of gel electrophoresis.
  • This jump in the pH value can in particular be 1.5 to 2.5, ie approximately 2, and in particular be carried out from pH 6.5 to pH 7.0 to pH 8.5 to pH 9.0, for example of pH 6 , 8 to pH 8.8.
  • the functional differences between collecting gel and separating gel can also be achieved in other ways than by the jump in the pH.
  • neutral SDS-polyacrylamide gels according to Graham et al., Proteomics 2005, 5, 2309-2314 differ in the concentrations of bis-tris-HCl and acrylamide, but not in the pH-value, which in principle also on polyacrylamide gel electrophoresis in the Presence of sarcosyl should be transferable.
  • a kit according to the invention for separating carboxyterminal variants of Abeta peptides by gel electrophoresis using the method according to the invention comprises at least one collection gel and one separating gel which have sarcosyl.
  • the kit may comprise a sarcosyl-containing cathode buffer and / or a sarcosyl-containing anode buffer.
  • the sarcosyl can be present in a concentration of between 0.1% and 0.8% (w / v) in the collection gel and / or in the separation gel and / or in the cathode buffer and / or the anode buffer of the kit.
  • the gels and buffers of the kit, including a sample buffer can be free of functional urea, which is particularly preferred.
  • the cathode buffer and / or the anode buffer of the kit may be a Tris-HEPES buffer, while the sample buffer of the kit may be a Tris-HCl buffer and the collection gel and / or the separation gel may be Tris-HCl polyacrylamide gels.
  • the sample buffer of the kit is typically sarcosyl-free and instead has SDS and / or LDS as the disintegrating detergent. Concretely, the SDS and / or LDS may be contained in a concentration of between 0.5% and 2% (w / v) in the sample buffer.
  • glycerol and / or sucrose may be contained in the sample buffer of the kit, in particular in a concentration between 5% and 10% (v / v) glycerol and / or in a concentration between 5% and 20% (w / v) sucrose) , Between the collection gel and the separating gel, a jump in the pH may be formed by 1.5 to 2.5 and / or from pH 6.6 to pH 7.0 to pH 8.5 to pH 9.0. Deviating from these statements, which focus on proven embodiments of the kit, there are various possible variations, as already mentioned in connection with the method according to the invention.
  • FIG. 1 schematically shows the performance of a gel electrophoresis according to the method according to the invention
  • FIG. 2 shows a kit according to the invention for carrying out the method according to the invention.
  • Fig. 1 shows schematically the performance of a gel electrophoresis.
  • sample pockets (see FIG. 2 BZ 10) are first rinsed in the upper end of a collecting gel 2 with a cathode buffer 7, and a cathode reservoir 6 adjoining the upper end of the collecting gel 2 is filled with cathode buffer 7.
  • At the collecting gel 2 closes down to a separating gel 3, which dips with its lower end in an anode buffer 4 in an anode reservoir 5.
  • an electric field 8 is applied across the collection gel 2 and the separation gel 3.
  • This electric field causes the Abeta peptides to move down through the gels 2, 3.
  • Different carboxyterminal variants of the abeta peptides move in the separation gel 3 at different rates because their electrophoretic mobility depends not only on their size but also on their charge carrier charge.
  • a stoichiometric loading of the carboxyterminal, hydrophobic regions of the Abeta peptides is dominant for their electrophoretic mobility, so that the carboxyl terminally longer variants move faster through the separating gel 3 than the carboxyterminal shorter variants. This is indicated in FIG.
  • the stoichiometric loading of the carboxy-terminal hydrophobic regions of the Abeta peptides with sarcosyl is achieved in the method according to the invention by incorporating into the collection gel 2 and into the separating gel 3 sarcosyl in a concentration of about 0.2% (w / v) sarcosyl and that at least the cathode buffer 7 contains sarcosyl in this concentration.
  • Sample buffer 1 contains no sarcosyl but SDS or LDS for the digestion of the Abeta peptides. It is understood that the illustrated in Fig. 1 bands of the various carboxy-terminal variants Aß1- 37, Aß1-38, Aß1-39, Aß1-40 and Aß1-42 only after their visualization, for example by color or fluorescent labeling, with a to all carboxy-terminal variants equally binding antibody show.
  • FIG. 2 shows a kit 9 according to the invention for carrying out the method according to the invention. This kit 9 has the collection gel 2 and the separating gel 3, which each contain sarcosyl. Here, in the upper region of the collecting gel 2 in the side view according to FIG.
  • kit 9 comprises sample buffer 1 containing SDS or LDS, and running buffer 1 1 containing sarcosyl, which can be used both as cathode buffer 7 and as anode buffer 4 according to FIG. EXEMPLARY EMBODIMENTS:
  • Cathode Buffer 100mM Tris- (hydroxymethyl) -aminomethane, 100m M H EPES, 0.25% (w / v) N-lauroyl sarcosine sodium salt
  • Anode buffer 100 mM Tris, 100 mM HEPES
  • the polymerization of acrylamide and bisacrylamide is started by adding ammonium persulfate and N, N, N ', N'-tetramethylethylenediamine. Immediately thereafter, the separating gel solution is mixed well and, using the BioRad Mini Protean Tetra Cell vertical gel electrophoresis system, poured between two 0.75 mm thick spacers to the desired height (about 5.5 cm) and 70 mm thick % 2-propanol overcoated.
  • the collection gel solution for 2 minigels (5 ml) is prepared, for example, according to the following pipetting scheme:
  • the concentration of acrylamide + bisacrylamide and the relative proportion of bisacrylamide (cross-linking degree) in the stacking gel can be varied.
  • the polymerization of the collecting gel is started by adding ammonium persulphate and TEMED (see above), the collecting gel solution is introduced between the glass plates and a sample comb introduced for shaping the sample bags. To polymerize the collection gel, the gel should be allowed to stand for at least one hour.
  • amyloid-ß peptide variants A ⁇ (1-37), Aß (1-38), Aß (1-39), Aß (1-40) and Aß (1- 42) amyloid-ß peptide variants A ⁇ (1-37), Aß (1-38), Aß (1-39), Aß (1-40) and Aß (1- 42) the use of a sarcosyl-polyacrylamide gel having a total concentration of 12% acrylamide + bisacrylamide (12% T) and a ratio of acrylamide to bisacrylamide of 19: 1 (5% C).
  • the total concentration of acrylamide plus bisacrylamide or u. U. modified the relative proportion of bisacrylamide (degree of crosslinking). Total concentrations of acrylamide + bisacrylamide between approx.
  • the samples are dissolved in a sample buffer containing sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS) and heated to 95 ° C for 5 minutes to denature the A ⁇ peptides.
  • SDS sodium dodecyl sulfate
  • LDS lithium dodecyl sulfate
  • the separation was carried out at a constant 160 V for about 40-55 minutes 100 mM tris- (hydroxymethyl) -aminomethane, 100 mM HEPES, 0.25% (w / v) N-lauroylsarcosine sodium salt as cathode buffer and 100 mM Tris , 100 mM HEPES as anodic buffer. It can be assumed that other electrophoresis buffers can also be used. detection:
  • Detection of the A ⁇ peptides can be accomplished by peptide / protein staining in the gel (e.g., Coomassie staining or silver staining or immunological Western blotting.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
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  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
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Abstract

La présente invention concerne la réalisation d'une électrophorèse sur gel en présence de lauroyl-sarcosinate de sodium (sarkosyl) pour séparer des variantes carboxy-terminales de peptides bêta-amyloïdes. Le sarkosyl peut être présent à une concentration comprise entre 0,1 % et 0,8 % (p/v) dans un gel d'empilement (2) et/ou dans un gel de séparation (3) et/ou dans un tampon cathodique (7) et/ou dans un tampon anodique (4) de l'électrophorèse sur gel.
PCT/EP2018/084629 2017-12-18 2018-12-12 Procédé pour séparer différents peptides bêta-amyloïdes par électrophorèse sur gel Ceased WO2019121263A1 (fr)

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DE102017130356.1A DE102017130356A1 (de) 2017-12-18 2017-12-18 Verfahren zur Auftrennung verschiedener Abeta-Peptide durch Gelelektrophorese
DE102017130356.1 2017-12-18

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110133091A (zh) * 2019-04-29 2019-08-16 中国科学院武汉物理与数学研究所 05sar-page及其制备方法和应用
US20220050078A1 (en) * 2019-04-29 2022-02-17 Innovation Academy For Precision Measurement Science And Technology, Chinese Academy Of Sciences Gel, marker, and kit for protein electrophoresis, and application of gel

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113311053A (zh) * 2021-06-29 2021-08-27 中国科学院精密测量科学与技术创新研究院 一种蛋白质电泳用凝胶、标记物、应用、及试剂盒

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WO2003096023A1 (fr) 2002-05-07 2003-11-20 Medical Research Council Solution tampon sarcosyl et utilisation associee pour la purification de la proteine prion
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WO2003096023A1 (fr) 2002-05-07 2003-11-20 Medical Research Council Solution tampon sarcosyl et utilisation associee pour la purification de la proteine prion
WO2004067561A1 (fr) * 2003-01-31 2004-08-12 Abbott Gmbh & Co. Kg Oligomeres de $g(b)(1-42) amyloides, derives de ces composes et anticorps destines a ceux-ci, procede de fabrication et utilisation de ces composes

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110133091A (zh) * 2019-04-29 2019-08-16 中国科学院武汉物理与数学研究所 05sar-page及其制备方法和应用
US20220050078A1 (en) * 2019-04-29 2022-02-17 Innovation Academy For Precision Measurement Science And Technology, Chinese Academy Of Sciences Gel, marker, and kit for protein electrophoresis, and application of gel

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