The resolution of AP in Bis-tris-HEPES-MES buffer system without stacking gel.

<p>The PAGE was carried out with a resolving gel of 9% in pH 6.5 Bis-tris−HEPES−MES buffer and with no stacking gel exclusively for the AP preparation. (a) the bands in natural colors, (b) the bands of native state in grey scale without red filter, (c) the bands of native state in grey scale with red filter, (d) the bands in fluorescence under UV-light at 365 nm and (e) the bands stained by Coomassie Blue G-250. The Tricine−Bis-tris electrode buffers in pH 6.3 were used in the PAGE. The sample used in the PAGE was the R-PC and AP fraction from the ion exchange chromatography.</p

Separation of Native Allophycocyanin and R-Phycocyanin from Marine Red Macroalga <i>Polysiphonia urceolata</i> by the Polyacrylamide Gel Electrophoresis Performed in Novel Buffer Systems

<div><p>Three buffer systems of Imidazole−Acetic acid, HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES were designed based on the principle of discontinuous polyacrylamide gel electrophoresis (PAGE) for the native PAGE which could be performed in pH 7.0 and 6.5 in order to analyze and prepare the minor components of allophycocyanin (AP) and R-phycocyanin (R-PC) from marine red macroalga <i>Polysiphonia urceolata</i>. These AP and R-PC phycobiliproteins are easily denatured in alkaline environments. The obtained results demonstrated that the PAGE modes performed in the buffer systems of HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES gave the satisfactory resolution and separation of AP and R-PC proteins. The absorption and fluorescence spectra of the AP and R-PC proteins which were prepared by the established PAGE modes proved that they maintained natural spectroscopic characteristics. The established PAGE modes may also provide useful references and selections for some other proteins that are sensitive to alkaline environments or are not effectively separated by the classical PAGE modes performed normally in alkaline buffer systems.</p></div

The resolution of R-PC and AP in Bis-tris-HEPES-MES buffer system with the stacking gel.

<p>The PAGE with the stacking gel of 4% in pH 6.0 Bis-tris−HEPES−Acetic acid buffer was performed respectively with: the resolving gel of 7% (A) and of 9.5% (B) in pH 7.0 (A) and pH 6.5 (B) Bis-tris−HEPES−MES buffers; the Tricine−Bis-tris electrode buffers in pH 6.5 (A) and pH 6.3 (B) were used in the PAGE. (a) the bands in natural colors, (b) the bands of native state in grey scale without red filter, (c) the bands of native state with red filter, (d) the bands in fluorescence under UV-light at 365 nm and (e) the bands stained by Coomassie Blue G-250. The sample used in the PAGE was the R-PC and AP fraction obtained by the ion exchange chromatography.</p

Absorption and fluorescence spectra of the R-PC and AP prepared by the PAGE in the HEPRES−Imidazole/Bis-tris and Bis-tris−HEPES−MES buffer systems.

<p>The absorption (solid line) and fluorescence (dot line) spectra of the prepared R-PCs and APs showed respectively in (A) and (B); the absorption spectrum of the sample, which was obtained by the ion exchange chromatography and used for the PAGE experiments, showed as dash lines both in (A) and (B). The absorption spectra were recorded in pH 7.0 phosphate buffers, and the fluorescence spectra of the R-PC and AP recorded in pH 7.0 phosphate buffer when excited respectively at 500 nm and 615 nm. The specific absorption and fluorescence bands of the R-PC (550 nm and 618 nm, and 574 nm and 640 nm), AP (653 nm and 620 nm, and 662 nm) and R-PE (499 nm and 570 nm) were labeled on the spectra.</p

The resolution of high molecular mass protein markers (native) by the PAGE in the three buffer systems.

<p>(A) Bis-tris−HEPES−MES buffer systems; (B) HEPES−Imidazole/Bis-tris buffer systems; (C) Imidazole−Acetic acid buffer systems. The PAGE was performed with a gradient resolving gel of 5%–20% in pH 7.0 and a stacking gel of 3% in pH 6.0. The band patterns of each PAGE (left) were visualized by Coomassie Blue G-250.</p

The native PAGE of the R-PC and AP sample performed in Tris−Gly (A), Tris−Barbital (B), Imidazole−Acetic acid (C) and Bis-tris−HEPES−MES (D) buffer systems.

<p>(A) the Tris−Gly PAGE with a resolving gel of 6.5% in pH 8.8 Tris−HCl buffer, a staking gel of 3% in pH 6.8 Tris−HCl buffer and an electrode buffer of Tris−Gly in pH 8.3; (B) the Tris−Barbital PAGE with a resolving gel of 6.5 in pH 7.5 Tris−HCl buffer, a staking gel of 3% in pH 5.5 Tris−HCl buffer and an electrode buffer of Tris−Barbital in pH 7.0; (C) the Imidazole−Acetic acid PAGE with a resolving gel of 6.5% and a staking gel of 3.0% in pH 6.5 and 5.4 Imidazole−Acetic acid buffers and an electrode buffer of Tricine−Imidazole in pH 6.3; (D) the Bis-tris−HEPES−MES PAGE with a resolving gel of 7.0% in pH 6.5 Bis-tris−HEPES−MES buffer, a stacking gel of 4.0% in pH 6.0 Bis-tris−HEPES buffer and electrode buffers of Bis-tris−Tricine and Bis-tris−Tris in pH 6.3. Protein band patterns were visualized in grey scale without (a) and with (b) red filter, in fluorescence (c) under UV-light at 360 nm and by Coomassie Blue G-250 staining (d). The used sample was the R-PC and AP fraction from the ion exchange chromatography, and the same amount of the sample was equally loaded on each mode of the PAGE.</p

The resolution of high and low molecular mass protein markers (SDS) by the PAGE in the three buffer systems.

<p>(A) Bis-tris−HEPES−MES buffer systems; (B) HEPES−Imidazole/Bis-tris buffer systems; (C) Imidazole−Acetic acid buffer systems. The PAGE was performed with a gradient resolving gel of 5%–20% in pH 7.0 and a stacking gel of 3% in pH 6.0, and 0.1% SDS was added in cathode buffers. The band patterns of each PAGE were visualized by Coomassie Blue G-250.</p

The native PAGE of the R-PC and AP sample performed in the HEPES−Imidazole buffer.

<p>The PAGE had a resolving gel of 7% in pH 6.5 HEPES−Imidazole buffer and a stacking gel of 4% in pH 5.4 Bis-tris−Acetic acid buffer; the Tricine−Bis-tris electrode buffers in pH 6.3 were employed as anode and cathode solutions. (a) the bands in natural colors, (b) the bands of native state in grey scale without red filter, (c) the bands of native state in grey scale with red filter and (d) the bands in fluorescence under UV-light at 365 nm. The R-PC and AP sample used in the PAGE was the R-PC fraction from the ion exchange chromatography.</p

The native PAGE of the purified R-PE (A) and the R-PC sample (B) performed in HEPES−Imidazole and Imidazole−Acetic acid buffer systems.

<p>The PAGE with the resolving gel of 9% in pH 6.5 and the stacking gel of 4% in pH 5.4 was performed respectively in: (a) and (c) HEPES−Imidazole buffers; (b) and (d) Imidazole−Acetic acid buffers. Imidazole−Acetic acid and Tricine−Imidazole buffers in pH 6.3 were employed respectively as anode and cathode solutions. The protein bands were visualized under UV-light (a and b) at 360 nm and by Coomassie Blue G-250 staining (c and d). D-PBP represented the dissociated phycobiliprotein. The R-PC sample used in the PAGE was the R-PC fraction obtained by the ion exchange chromatography.</p

Media 1: Equilibrium orientations and positions of non-spherical particles in optical traps

Originally published in Optics Express on 04 June 2012 (oe-20-12-12987