A Method for Enhancing the Sensitivity and Stability of Stains-All for Phosphoproteins Separated in Sodium Dodecyl Sulfate–Polyacrylamide Gels

Myers, Jody M.; Veis, Arthur; Sabsay, Boris; Wheeler, A. P.

doi:10.1006/abio.1996.0361

Cited by 36 publications

(18 citation statements)

References 2 publications

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“…1, lane 2), which is diagnostic of a high negative charge density (21). All other phosphoproteins exhibited basic isoelectric points as revealed by their quantitative adsorption to a cation-exchange resin at pH 7 (unpublished results).…”

Section: Resultsmentioning

confidence: 82%

Biosilica formation in diatoms: Characterization of native silaffin-2 and its role in silica morphogenesis

Poulsen

Sumper

Kröger

2003

Proc. Natl. Acad. Sci. U.S.A.

321

303

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The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100 -1000 nm, which is characteristic for diatom biosilica nanopatterns.T he biological formation of intracellular or extracellular skeletons made of amorphous hydrated SiO 2 (biosilica) is relatively frequent among the protists and also occurs in many higher plants (1, 2). To date the organic molecules involved in biosilica formation have mainly been studied in sponges and diatoms (3). From the silica spicules of the sponge Thetya aurantia, proteaselike proteins termed silicateins were characterized that catalyze silica formation from tetraethoxysilane in vitro (4, 5). Diatoms are unicellular algae that have the extraordinary capability to produce an enormous variety of biosilica structures (6). Each diatom species is characterized by a specific biosilica cell wall that contains regularly arranged slits or pores in the size range between 10 and 1,000 nm (nanopatterned biosilica). Biosilica morphogenesis takes place inside the diatom cell within a specialized membrane-bound compartment termed the silica deposition vesicle (SDV). It has been postulated that the SDV contains a matrix of organic macromolecules that not only regulate silica formation but also act as templates to mediate biosilica nanopatterning (7-9). Insight into the nature of this organic matrix has been gained through the characterization of diatom biosilica-associated peptides (silaffins) and long-chain polyamines (LCPA), both of which accelerate silica formation from a silicic acid solution in vitro (10-12). Although native silaffin-1A (natSil-1A) and LCPA, respectively, mediate the formation of unusual silica structures in vitro (networks of irregularly shaped silica bands and large spherical silica particles) (11, 12), none of these are akin to diatom biosilica nanopatterns.Recently, it was shown that phosphorylation of natSil-1A is essential for silica formation activity (12), leading to the speculation that phosphorylated components may be generally important for biosilica morphogenesis. Therefore, a search for additional silica-associated phosphoproteins, which are able...

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Section: Resultsmentioning

confidence: 82%

Biosilica formation in diatoms: Characterization of native silaffin-2 and its role in silica morphogenesis

Poulsen

Sumper

Kröger

2003

Proc. Natl. Acad. Sci. U.S.A.

321

303

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“…Neither protein band was seen following staining with Coomassie brilliant blue, as these highly acidic, phosphorylated [0]proteins react poorly or do not react at all with Coomassie brilliant blue. 46 The identities of OPN and BSP were further confirmed by Western immunoblots using antibodies specific to OPN and BSP, respectively (data not shown).…”

Section: Opn and Bsp Isolationmentioning

confidence: 83%

MC3T3-E1 cell adhesion to hydroxyapatite with adsorbed bone sialoprotein, bone osteopontin, and bovine serum albumin

Bernards

Qin

Jiang

2008

Colloids and Surfaces B: Biointerfaces

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Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.

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“…"Stains-all" staining with the cationic carbocyanine dye [16,17] was performed for the detection of such molecules within the isolated ␤-lactoglobulin fractions. However, no blue or purple staining specific for such phosphorylated proteins could be detected within the respective fractions (not shown).…”

Section: Resultsmentioning

confidence: 99%

Improved purification of β-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

Schlatterer

Baeker²,

Schlatterer

2004

Journal of Chromatography B

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A Method for Enhancing the Sensitivity and Stability of Stains-All for Phosphoproteins Separated in Sodium Dodecyl Sulfate–Polyacrylamide Gels

Cited by 36 publications

References 2 publications

Biosilica formation in diatoms: Characterization of native silaffin-2 and its role in silica morphogenesis

Biosilica formation in diatoms: Characterization of native silaffin-2 and its role in silica morphogenesis

MC3T3-E1 cell adhesion to hydroxyapatite with adsorbed bone sialoprotein, bone osteopontin, and bovine serum albumin

Improved purification of β-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

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