QPNC-PAGE

QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis ("quantitative native PAGE"), is a high-resolution and a highly accurate technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point. This standardized variant of native gel electrophoresis is used by biologists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins or protein isoforms in complex protein mixtures.[1] The high reproducibility and the high-yield electroelution (> 95%) of proteins performed by this technique strongly correlates with the polymerization time (69 hr) of the acrylamide (AA) gels.

As omics platform for quantitative biomedical approaches QPNC-PAGE contributes to the development of metal-based drugs for protein-misfolding diseases,[2] and as such, to the emerging field of biobased economy. Another important link addresses the investigation of the role of environmental contaminants like copper in the etiology of Alzheimer's disease (AD)[3] because inorganic copper[4] that cannot be detoxified completely by the liver, may be a major triggering agent in AD.[5]

Introduction

Proteins perform several functions in living organisms, including catalytic reactions and transport of molecules or ions within the cells, the organs or the whole body. The understanding of the processes in organisms, which are driven by chemical reactions, depends to a great extent on our ability to isolate active proteins in biological samples for more detailed examination of chemical structure and physiological function. As about 30-40% of all known proteins contain one or more metal ion cofactors (e.g., ceruloplasmin), especially native metalloproteins have to be isolated, identified and quantified in biomatrices. Many of these cofactors play a key role in vital enzymatic catalytic processes or stabilize globular protein molecules.[6] Therefore, the electrophoresis and other separation techniques are highly relevant as initial step of protein analysis followed by mass spectrometric and magnetic resonance methods for quantifying and identifying the proteins of interest.

In this context, QPNC-PAGE is introduced as part of a combined procedure for biomedical approaches to investigate protein-protein interactions and protein-cofactor interactions in clinical samples. A major goal of this article is to elucidate the relationship between the polymerization time of acrylamide gels, gel stability and analytical results obtained by the following procedure.

Method

Separation and buffering mechanisms

Equipment for preparative electrophoresis: electrophoresis chamber, peristaltic pump, fraction collector, buffer recirculation pump and UV detector (in a refrigerator), power supply and recorder (on a table).

In gel electrophoresis proteins are normally separated by charge and size. The aim of isoelectric focusing (IEF) is to separate proteins according to their isoelectric point (pI), thus, according to their charge at different pH values.[7] Here, the same mechanism is accomplished in a commercially available electrophoresis chamber (see figure Equipment) for separating charged biomolecules, for example, superoxide dismutase (SOD)[8] or allergens,[9] at continuous pH conditions and different velocities of migration depending on different isoelectric points.[10]

Due to the specific properties of the prepared gel and electrophoresis buffer solution (which is basic and contains Tris-HCl and NaN3), most proteins of a biological system are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field. At the anode, electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions. The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water. Thus, the Tris-based buffering mechanism causes a constant pH in the buffer system.[11]

Although the pH value (10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the separated ring-shaped protein bands are eluted continuously into a physiological buffer solution (pH 8.00) and isolated in different fractions (see figure Electropherogram). Provided that irreversible denaturation cannot be demonstrated (by an independent procedure), most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C.[12] As the Joule heat generated during electrophoresis may have a negative impact on the stability and migration behavior of proteins, the separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator at 4 °C (see figure Equipment).

Gel properties and polymerization time

Best polymerization conditions are obtained at 25-30 °C[13] and polymerization seems terminated after 20-30 min of reaction although residual monomers (10-30%) are detected after this time.[14] The time of polymerization of the gel may directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression of the gels and their pores with the longer incubation times (see figure Electropherogram). In order to insure maximum reproducibility in gel pore size and to obtain a fully polymerized and non-restrictive large pore gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature (RT). After this time the gel is in its lowest energy state and ready to be run. The exothermic heat generated by the polymerization processes is dissipated constantly. As a result, the prepared gel is homogeneous, inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins.[15]

The 4% T, 2.67% C gel is pre-run to equilibrate it. It is essentially non-sieving and optimal for electrophoresis of proteins that are smaller and larger than 200 ku (cf. agarose gel electrophoresis). Proteins migrate in it more or less on the basis of their free mobility.[16] For these reasons interactions of the gel with the biomolecules are negligibly low and the proteins separate cleanly and predictably (see figure Electropherogram). The separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides, metallothionein) are not dissociated into apoproteins and metal cofactors.[17]

Reproducibility and recovery

Electropherogram showing four PAGE runs of a native chromatographically prepurified high molecular weight cadmium protein (≈ 200 ku) as a function of time of polymerization of the gel (4% T, 2.67% C). Detection method for Cd cofactors (in µg/L): GF-AAS

The bioactive structures (native or 3D conformation or shape) of the isolated protein molecules do not undergo any significant conformational changes. Thus, active metal cofactor-containing proteins can be isolated reproducibly in the same fractions after a PAGE run (see figure Electropherogram). A shifting peak in the respective electropherogram may either indicate that a denatured metalloprotein is available in a complex protein mixture to be separated or the standardized time of gel polymerization (69 hr, RT) is not implemented in a PAGE experiment. A lower deviation of this standardized polymerization time (< 69 hr) stands for incomplete polymerization, whereas exceeding this time limit (> 69 hr) is an indicator of gel aging (see figure Electropherogram). Under standard conditions metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a quantitative yield of more than 95%.[18]

By preparative SDS polyacrylamide gel electrophoresis standard proteins (cytochrome c, aldolase, ovalbumin and bovine serum albumin) with molecular masses of 14–66 ku can be recovered with an average yield of about 73,6%.[19] Preparative isotachophoresis (ITP) is applied for isolating palladium-containing proteins with molecular masses of 362 ku (recovery: 67%) and 158 ku (recovery: 97%).[20]

Quantification and identification

Low concentrations (ppb-range) of Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and absolutely quantified in an aliquot of a fraction by inductively coupled plasma mass spectrometry (ICP-MS)[21] or total reflection X-ray fluorescence (TXRF),[22] for example. In case of ICP-MS the structural information of the associated metallobiomolecules[23] is irreversibly lost due to ionization of the sample with plasma.[24] Because of high purity and optimized concentration of the separated metalloproteins, for example, therapeutic recombinant plant-made pharmaceuticals such as copper chaperone for superoxide dismutase (CCS) from medicinal plants, in a few specific PAGE fractions, the related structures of these analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions.[25]

Applications

Improperly folded metal proteins, for example, CCS or Cu/Zn-superoxide dismutase (SOD1) present in brain, blood or other clinical samples, are indicative of neurodegenerative diseases like Alzheimer's disease or Amyotrophic Lateral Sclerosis (ALS).[26] Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ions (e.g., Cu1+/2+, Zn2+, Fe2+/3+, Mn2+, Ni3+) in organisms, and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm. Otherwise, transition metal ions take part in Fenton-like reactions in which deleterious hydroxyl radical is formed, which unrestrained would be destructive to proteins.[27] The loss of (active) CCS increases the amyloid-β production in neurons which, in turn, is a major pathological hallmark of AD.[28] Therefore, copper chaperone for superoxide dismutase is proposed to be one of the most promising biomarkers of Cu toxicity in these diseases.[29] CCS should be analysed primarily in blood because a meta-analysis of serum data showed that AD patients have higher levels of serum Cu than healthy controls.[30]

Quantitative native PAGE is applied in the field of molecular biology to purify enzymes and recombinant proteins of microbial strains.[31] The thermostability and activity of enzymes expressed by thermophilic bacteria is genetically encoded. In the frame of bioeconomy these biomolecules can be used as research reagents and as catalysts for industrial processes.[32]

Principle

History

In the 20th century it was generally accepted that ammonium persulfate (APS)/TEMED-initiated reactions should be allowed to proceed for 5-15 min[33] to approx. 1-2 hr[34] to ensure maximum reproducibility in gel pore size of PAGE gels. In another review it is recommended to allow the gel to polymerize overnight at room temperature.[35] Longer incubation times (16 hr to 2 wk) to finish the matrix formation might not have any essential effects on the protein separation process.[36] Explicit polymerization times for specific applications or methods are not mentioned in the literature.[37] Being hydrolyzed into polyacrylic acid polyacrylamide gels were further considered as inherently unstable with respect to polymer resistance to alkaline media.[38] Under these conditions only a semiquantitative characterization of metalloproteins was enabled.[39] Therefore, the relationship between the polymerization time, gel stability and analytical results had to be investigated addressing the absolute quantification of specific protein molecules:

In 2001 a new basic principle of gel electrophoresis was discovered at the Forschungszentrum Jülich, applied for patent in 2003 and subsequently issued in 2014.[40] This invention provided the first conclusive evidence that the time of polymerization of a polyacrylamide gel directly affects the result of protein purification because the separation properties implying the mechanical and chemical stability of a gel and its pores are determined by this parameter. As a consequence, the peak-elution times of the separated metalloproteins in the electropherogram may vary considerably (see figure Electropherogram). On the other hand, the results of protein electrophoresis can be optimized in terms of reliability and avoiding artifacts by strict adherence to a standardized time of polymerization (69 hr, RT) for acrylamide gels implying inherent stability at basic pH. This optimization process of selected electrophoretic parameters paved the way to "quantitative native PAGE" by the Jülicher researcher Bernd Kastenholz.[41]

Although the quality, quantity and mixing ratio of TEMED, APS and AA/Bis-AA as well as the ambient temperature are the most important factors to initiate and impel the polymerization reaction of PAGE gels, it is evident that the time of polymerization is the limiting factor in these chemical processes. Systematic investigations of the gel stability over time reveal significant changes in gel structure by day 3 (72 hr) after polymerization (5-15 min).[42] These results are in excellent agreement with the finding that the gel aging begins after 69 hr gelation time, as reported here. Given these new findings a re-assessment of past results and published data related to protein electrophoresis[43] and quantitative proteome analysis is necessary.[44]

Persons

First publications (2009) concerning the medical applications of this technique were edited or co-authored by the Münsteran human geneticist Prof. Jürgen Horst and the well-known American scientist and internationally recognized expert in the fields of DNA sequence analysis and protein electrophoresis David E. Garfin. In the 1970s and the early 1980s Dr. Garfin became one of the most important investigators and early pioneers in the field of prion research (scrapie) in the team of Stanley B. Prusiner at the UC SF.[45] In addition to his pioneering work on one- and two-dimensional gel electrophoresis in the 1990s at Bio-Rad Laboratories[46] he became co-editor of the Handbook of Isoelectric Focusing and Proteomics (2005)[47] and co-authored a worth reading chapter on bioseparation methods in the Kirk-Othmer Encyclopedia of Chemical Technology (2007).[48] For significant contributions to electrophoresis in both engineering and biology communities Dave Garfin received the 2013 AES Electrophoresis Society Career Award in San Francisco.[49]

Biomedicine

Said authors anticipated that the above-mentioned approach might be implemented in the therapy and diagnosis of several protein-misfolding diseases: on the one hand, copper chaperone for superoxide dismutase may serve as a biomarker for Cu toxicity in neurodegenerative diseases, on the other hand, copper chaperones[50] and other non-proteinogenic metallopharmaceuticals (e.g., Cu orotate,[51] Li chloride[52]) are indicated as lead compounds for the etiological treatment of Alzheimer's disease.[53]

As the mis-localization of metal ions (in particular Cu[54]) in the cell is most likely responsible for the onset and progression of sporadic and genetic forms of Alzheimer's disease and other dementias, said compounds may pass the blood brain barrier and trigger a metal-mediated signaling cascade of biochemical reactions that restore and maintain metal homeostasis in order to preserve the neuronal function in the brain of AD patients.[55] As cellular responses to these reactions the production of amyloid-β peptides and oxidative processes are normalized and neuritic plaques of AD brains are degraded by upregulation of the proteasome and other molecular mechanisms.[56] In these processes, especially protein-protein interactions play a crucial role. In general, the relative biochemical impact of an applied metal-containing compound (metal-based drug) is depending on its dose, bioavailability, trace metal binding form (chemical form) and accuracy of quantitative measurement.[57]

According to the Hofmeister series salt effects could be another approach for the treatment of Alzheimer's disease by using certain salts for dissolving protein aggregates or inhibiting amyloid formation.[58] Adding salts to complex protein mixtures, however, may induce a shift of isoelectric points, and thus, affect protein structure and activity. In this context, one study found that kosmotropic salts (e.g., ammonium sulfate) that were used for the precipitation of native proteins in solution, subsequently may result in denatured high molecular weight metalloproteins in the same solution.[59] It can be concluded that ions which promote aggregation, simultaneously cause denaturation of the native conformation. In opposite case chaotropic salts force globular proteins to unfold.[60] For these reasons, restoring and maintaining the physiological states of aggregated or unfolded (metal) proteins by applying Hofmeister salts in the therapy of Alzheimer's disease is very unlikely to happen. For example, Li chloride caused a reduction in protein synthesis and hence the level of amyloid-β peptides, however, this compound may also generate severe side effects induced by long-term, high-dose lithium.[61]

Curcumin is one of the most promising therapeutic agents for inflammation, cystic fibrosis, Alzheimer's disease and cancer because these biomolecules scavenge radicals and maintain the levels of (active) antioxidant enzymes (e.g., SOD1)[62] in the presence of copper. Curcumin is almost free from side effects, however, limited for application due to its poor bioavailability.[63] The development and production of plant-made pharmaceuticals (e.g., CCS) by combining modern biochemical techniques and plant phenotyping platforms,[64] may help to provide bioactive therapeutic proteins as a major basis for pharmacological efficiency in conformational diseases. Said molecules are non-toxic, reveal a high specificity and their mechanisms of action are well known in living organisms.[65]

Conclusions

High protein yield and purity are the bottleneck of quantitative native protein analysis in biological samples. QPNC-PAGE is a unique method and the initial step that opens the bottleneck of protein isolation in complex protein mixtures. This preparative technique is based on a new principle and a new constant of acrylamide gel electrophoresis implying the accurate control of gel pore size and stability by the time of polymerization of acrylamide. A combined procedure of solution NMR, QPNC-PAGE, and ICP-MS may be the key for the diagnosis and therapy of several protein-misfolding diseases related to dyshomeostasis of biometal metabolism in the human brain. Biological proteins possess the pharmacological potential to restore and maintain the homeostasis of metal ions in conformational diseases.

See also

References

  1. ↑ Seelert H, Krause F (2008). "Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels". Electrophoresis 29 (12): 2617–36. doi:10.1002/elps.200800061. PMID 18494038.
  2. ↑ Aderibigbe, BA (2015). "Polymeric prodrugs containing metal-based anticancer drugs". Journal of Inorganic and Organometallic Polymers and Materials 25 (25): 339–53. doi:10.1007/s10904-015-0220-7.
  3. ↑ Yegambaram M, Manivannan B, Beach TG, Halden RU (2015). "Role of environmental contaminants in the etiology of Alzheimer's disease: a review". Current Alzheimer Research 12 (2): 116–46. doi:10.2174/1567205012666150204121719. PMC 4428475. PMID 25654508.
  4. ↑ Brewer GJ (2011). "Issues raised involving the copper hypotheses in the causation of Alzheimer's Disease". International Journal of Alzheimer’s Disease 2011: 1–11. doi:10.4061/2011/537528. PMC 3172975. PMID 21922048.
  5. ↑ Brewer GJ (2015). "Divalent copper as a major triggering agent in Alzheimer’s Disease". Journal of Alzheimer's disease 46 (3): 593–604. doi:10.3233/JAD-143123. PMID 25854930.
  6. ↑ Finney LA, O'Halloran TV (2003). "Transition metal speciation in the cell: insights from the chemistry of metal ion receptors". Science 300 (5621): 931–6. doi:10.1126/science.1085049. PMID 12738850.
  7. ↑ Garfin DE (1990). "Isoelectric focusing". Methods in Enzymology 182: 459–77. doi:10.1016/0076-6879(90)82037-3. PMID 2314254.
  8. ↑ Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996). "A novel nickel-containing superoxide dismutase from Streptomyces spp". Biochemical Journal 318 (Pt 3): 889–96. PMC 1217701. PMID 8836134.
  9. ↑ Suck R, Petersen A, Weber B, Fiebig H, Cromwell O (2004). "Analytical and preparative native polyacrylamide gel electrophoresis: investigation of the recombinant and natural major grass pollen allergen Phl p 2". Electrophoresis 25 (1): 14–9. doi:10.1002/elps.200305697. PMID 14730563.
  10. ↑ McLellan T (1982). "Electrophoresis buffers for polyacrylamide gels at various pH". Analytical Biochemistry 126 (1): 94–9. doi:10.1016/0003-2697(82)90113-0. PMID 7181120.
  11. ↑ Kastenholz B (2006). "Comparison of the electrochemical behavior of the high molecular mass cadmium proteins in Arabidopsis thaliana and in vegetable plants on using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE)". Electroanalysis 18 (1): 103–6. doi:10.1002/elan.200403344.
  12. ↑ Gordon AH (1969). "Electrophoresis of proteins in polyacrylamide and starch gels (Part I, Chapter 2 Acrylamide gel)". Laboratory Techniques in Biochemistry and Molecular Biology 1: 34–45. doi:10.1016/S0075-7535(08)70324-3.
  13. ↑ Gelfi C, Righetti PG (1981). "Polymerization kinetics of polyacrylamide gels II. Effect of temperature". Electrophoresis 2: 220–28. doi:10.1002/elps.1150020405.
  14. ↑ Chen B, Chrambach A (1979). "Estimation of polymerization efficiency in the formation of polyacrylamide gel, using continuous optical scanning during polymerization". Journal of Biochemical and Biophysical Methods 1 (2): 105–16. doi:10.1016/0165-022X(79)90017-4. PMID 551105.
  15. ↑ Garfin DE (2009). "25th Annual Meeting of the American Electrophoresis Society". Expert Review of Proteomics 6 (3): 239–41. doi:10.1586/epr.09.18. PMID 19489696.
  16. ↑ Garfin DE (2009) [1990]. "Chapter 29 one-dimensional gel electrophoresis". Methods in Enzymology 463: 497–513. doi:10.1016/S0076-6879(09)63029-9. ISBN 978-0-12-374536-1. PMID 19892189.
  17. ↑ Fitri N, Kastenholz B, Buchari B, Amran MB, Warganegara FM (2008). "Molybdenum speciation in raw phloem sap of castor bean". Analytical Letters 41 (10): 1773–84. doi:10.1080/00032710802162442.
  18. ↑ Kastenholz B (2006). "Important contributions of a new quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) procedure for elucidating metal cofactor metabolisms in protein-misfolding diseases – a theory". Protein and Peptide Letters 13 (5): 503–8. doi:10.2174/092986606776819637. PMID 16800806.
  19. ↑ Ohhashi T, Moritani C, Andoh H, Satoh S, Ohmori S, Lottspeich F, Ikeda M (1991). "Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies". Journal of Chromatography 585 (1): 153–9. PMID 1666109.
  20. ↑ Weber G, Messerschmidt J, von Bohlen A, Kastenholz B, Günther K (2004). "Improved separation of palladium species in biological matrices by using a combination of gel permeation chromatography and isotachophoresis". Electrophoresis 25 (12): 1758–64. doi:10.1002/elps.200305833. PMID 15213973.
  21. ↑ Rašovský P (2011). "Utilisation of column gel eletrophoresis for on-line connection to ICP-MS for metalloproteomics". Archive of Thesis, Masaryk University, Brno.
  22. ↑ Pessanha S, Carvalho ML, Becker M, von Bohlen A (2010). "Quantitative determination on heavy metals in different stages of wine production by total reflection X-ray fluorescence and energy dispersive X-ray fluorescence: comparison on two vineyards". Spectrochimica Acta Part B Atomic Spectroscopy 65 (6): 504–7. doi:10.1016/j.sab.2010.04.003.
  23. ↑ Jakubowski N, Lobinski R, Moens L (2004). "Metallobiomolecules. The basis of life, the challenge of atomic spectroscopy". Journal of Analytical Atomic Spectrometry 19 (1): 1–4. doi:10.1039/B313299B.
  24. ↑ Mounicou S, Szpunar J, Lobinski R (2009). "Metallomics: the concept and methodology". Chemical Society Reviews 38 (4): 1119–38. doi:10.1039/B713633C. PMID 19421584.
  25. ↑ Kastenholz B, Garfin DE (2009). "Medicinal plants: a natural chaperones source for treating neurological disorders". Protein and Peptide Letters 16 (2): 116–20. doi:10.2174/092986609787316234. PMID 19200033.
  26. ↑ Schümann K, Classen HG, Dieter HH, König J, Multhaup G, Rükgauer M, Summer KH, Bernhardt J, Biesalski HK (2002). "Hohenheim consensus workshop: copper". European Journal of Clinical Nutrition 56 (6): 469–83. doi:10.1038/sj.ejcn.1601315. PMID 12032645.
  27. ↑ Robinson NJ, Winge DR (2010). "Copper metallochaperones". Annual Review of Biochemistry 79: 537–62. doi:10.1146/annurev-biochem-030409-143539. PMID 20205585.
  28. ↑ Gray EH, De Vos KJ, Dingwall C, Perkinton MS, Miller CC (2010). "Deficiency of the copper chaperone for superoxide dismutase increases amyloid-β production". Journal of Alzheimer's Disease 21 (4): 1101–5. doi:10.3233/JAD-2010-100717. PMID 20693630.
  29. ↑ Pal A (2014). "Copper toxicity induced hepatocerebral and neurodegenerative diseases: an urgent need for prognostic biomarkers". Neurotoxicology 40: 97–101. doi:10.1016/j.neuro.2013.12.001. PMID 24342654.
  30. ↑ Bucossi S, Ventriglia M, Panetta V, Salustri C, Pasqualetti P, Mariani S, Siotto M, Rossini PM, Squitti R (2011). "Copper in Alzheimer's disease: a meta-analysis of serum, plasma, and cerebrospinal fluid studies". Journal of Alzheimer's Disease 24 (1): 175–85. doi:10.3233/JAD-2010-101473. PMID 21187586.
  31. ↑ BIOMIG. Biotransformations & Microbial Genetics Laboratory (Tezel U), Institute of Environmental Sciences, Boğaziçi University, Instanbul.
  32. ↑ Vieille C, Zeikus GJ (2001). "Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability" (PDF). Microbiology and Molecular Biology Reviews 65 (1): 1–43. doi:10.1128/MMBR.65.1.1-43.2001. PMC 99017.
  33. ↑ Chrambach A, Rodbard D (1971). "Polyacrylamide gel electrophoresis". Science 172 (3982): 440–51. doi:10.1126/science.172.3982.440. PMID 4927678.
  34. ↑ Wittig I, Braun H-P, Schägger H (2006). "Blue native PAGE". Nature Protocols 1 (1): 418–28. doi:10.1038/nprot.2006.62. PMID 17406264.
  35. ↑ Kurien BT, Scofield RH (2012). "Common artifacts and mistakes made in electrophoresis". Methods in Molecular Biology 869: 633–40. doi:10.1007/978-1-61779-821-4_58. PMID 22585529.
  36. ↑ Kozulic B (1995). "Gel composition in gels for submurged gel electrophoresis". Patent US5458760 (A).
  37. ↑ Westermeier R (2005). Electrophoresis in practice: a guide to methods and applications of DNA and protein separations, fourth edition. ISBN 9783527311811. Wiley. doi:10.1002/3527603468.
  38. ↑ Boschetti E (1989). "Polyacrylamide derivatives to the service of bioseparations". Journal of Biochemical and Biophysical Methods 19 (1): 21–36. doi:10.1016/0165-022X(89)90048-1. PMID 2809065.
  39. ↑ Günther K, Ji G, Kastenholz B (2000). "Characterization of high molecular weight cadmium species in contaminated vegetable food". Fresenius' Journal of Analytical Chemistry 368 (2): 281–7. doi:10.1007/s002160000431. PMID 11220593.
  40. ↑ Kastenholz B (2014) [2003]. "Method for isolating metal cofactors out from biologically-organic systems involving the use of preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE)". Patent WO2005014147 (A2).
  41. ↑ Kastenholz B (2004). "Preparative native continuous polyacrylamide gel electrophoresis (PNC‐PAGE): an efficient method for isolating cadmium cofactors in biological systems". Analytical Letters 37 (4): 657–65. doi:10.1081/AL-120029742.
  42. ↑ Denisin AK, Pruitt BL (2016). "Tuning the range of polyacrylamide gel stiffness for mechanobiology applications". ACS Applied Materials & Interfaces. doi:10.1021/acsami.5b09344. PMID 26816386.
  43. ↑ Garfin DE (2003). "Chapter 7 gel electrophoresis of proteins". Essential Cell Biology Vol 1 Cell Structure: 197–268. ISBN 9780199638307.
  44. ↑ Aebersold R (2003). "Quantitative proteome analysis: methods and applications". The Journal of Infectious Diseases 187 (Supplement 2): S315–20. doi:10.1086/374756.
  45. ↑ Prusiner SB, Garfin DE, Cochran SP, McKinley MP, Groth DF, Hadlow WJ, Race RE, Eklund CM (1980). "Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent". Journal of Neurochemistry 35 (3): 574–82. doi:10.1111/j.1471-4159.1980.tb03693.x. PMID 6778963.
  46. ↑ Garfin DE (2003). "Two-dimensional gel electrophoresis: an overview" (PDF). TrAC Trends in Analytical Chemistry 22 (5): 263–72. doi:10.1016/S0165-9936(03)00506-5.
  47. ↑ Garfin DE, Ahuja S (2005). "Handbook of Isoelectric Focusing and Proteomics". Separation Science and Technology 7: 1–334. ISBN 978-0-12-088752-1.
  48. ↑ Garfin DE, Ahuja S (2007). "Bioseparations, separation methods in proteomics". Kirk-Othmer Encyclopedia of Chemical Technology: 1–49. doi:10.1002/0471238961.biosahuj.a01.
  49. ↑ Fintschenko Y, Salmanzadeh A, Davalos R (2014). "AES 2013: Annual Meeting of the AES Electrophoresis Society". American Laboratory.
  50. ↑ Markossian KA, Kurganov BI (2003). "Copper chaperones, intracellular copper trafficking proteins. Function, structure, and mechanism of action". Biochemistry (Moscow) 68 (8): 827–37. doi:10.1023/A:1025740228888. PMID 12948382.
  51. ↑ Kaden D, Bush AI, Danzeisen R, Bayer TA, Multhaup G (2011). "Disturbed copper bioavailability in Alzheimer's Disease". International Journal of Alzheimer’s Disease 2011: 1–5. doi:10.4061/2011/345614. PMC 3227474. PMID 22145082.
  52. ↑ Phiel CJ, Wilson CA, Lee VM, Klein PS (2003). "GSK-3α regulates production of Alzheimer's disease amyloid-β peptides". Nature 423 (6938): 435–9. doi:10.1038/nature01640. PMID 12761548.
  53. ↑ Kastenholz B, Garfin DE, Horst J, Nagel KA (2009). "Plant metal chaperones: a novel perspective in dementia therapy". Amyloid 16 (2): 81–3. doi:10.1080/13506120902879392. PMID 20536399.
  54. ↑ Squitti R, Polimanti R (2013). "Copper phenotype in Alzheimer’s disease: dissecting the pathway". American Journal of Neurodegenerative Disease 2 (2): 46–56. PMC 3703119. PMID 23844331.
  55. ↑ Sastre M, Ritchie CW, Hajji N (2015). "Metal ions in Alzheimer’s disease brain" (PDF). JSM Alzheimer’s Disease and Related Dementias 2 (1): 1014.
  56. ↑ Saibil H (2013). "Chaperone machines for protein folding, unfolding and disaggregation". Nature Reviews Molecular Cell Biology 14 (10): 630–42. doi:10.1038/nrm3658. PMID 24026055.
  57. ↑ Thompson KH, Orvig C (2003). "Boon and bane of metal ions in medicine". Science 300 (5621): 936–9. doi:10.1126/science.1083004. PMID 12738851.
  58. ↑ Yeh V, Broering JM, Romanyuk A, Chen B, Chernoff YO, Bommarius AS (2009). "The Hofmeister effect on amyloid formation using yeast prion protein". Protein Science 19 (1): 47–56. doi:10.1002/pro.281. PMID 19890987.
  59. ↑ Kastenholz B (2007). "New hope for the diagnosis and therapy of Alzheimer's disease". Protein and Peptide Letters 14 (4): 389–93. doi:10.2174/092986607780363970. PMID 17504097.
  60. ↑ Wang W, Roberts CJ (2010). Aggregation of therapeutic proteins. ISBN 978-0-470-41196-4. Wiley.
  61. ↑ Sofola-Adesakin O, Castillo-Quan JI, Rallis C, Tain LS, Bjedov I, Rogers I, Li L, Martinez P, Khericha M, Cabecinha M, Bähler J, Partridge L (2014). "Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease". Frontiers in Aging Neuroscience 6: 190. doi:10.3389/fnagi.2014.00190.
  62. ↑ Matés JM, Pérez-Gómez C, Núñez de Castro I (1999). "Antioxidant enzymes and human diseases". Clinical Biochemistry 32 (8): 595–603. doi:10.1016/S0009-9120(99)00075-2. PMID 10638941.
  63. ↑ Leung MH, Harada T, Kee TW (2013). "Delivery of curcumin and medicinal effects of the copper(II)-curcumin complexes". Current Pharmaceutical Design 19 (11): 2070–83. doi:10.2174/138161213805289237. PMID 23116313.
  64. ↑ Li L, Qin Z, Huang, D (2014). "A review of imaging techniques for plant phenotyping". Sensors 14: 20078–111. doi:10.3390/s141120078. PMC 4279472. PMID 25347588.
  65. ↑ Nagel KA, Kastenholz B, Gilmer F, Schurr U, Walter A (2010). "Novel detection system for plant protein production of pharmaceuticals and impact on conformational diseases". Protein and Peptide Letters 17 (6): 723–31. doi:10.2174/092986610791190282. PMID 20015023.

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