Albumin Blue

The helium-neon (HeNe) laser immediately comes to mind, having a very useful spectral line at 633 nm for steady-state red/near-IR fluorescence studies. Kessler and Wolfbeis have demonstrated the fluorescence assay of the protein human serum albumin using the probe albumin blue excited with a red HeNe laser.(71) Another useful wavelength available from the green HeNe laser is at 543.5 nm and this has been used with phase-modulation fluorometry by Lakowicz etal. to study probes such as carboxy seminaphtorhodafluor-6 (SNARF-6) as a means of measuring pH.(72)... 

Figure 15.3. Overlaid HSQC spectra of biopolymers on IHSS peat. (A) Biopolymers lignin (gray), amylopectin (red), albumin (blue) and cuticle (green) overlaid on each other. (B) All biopolymers are illustrated in black. (C) IHSS humic acid extract from peat. (D) Biopolymers (black) overlaid on IHSS peat (green). The highlighted areas in 2D are those not well represented by biopolymers in the HA, namely complex carbohydrates and p- ydroxybcnzoatcs from lignin [see Kelleher and Simpson (2006) for more details]. See color insert. Reprinted from Kelleher, B. R, and Simpson, A. J. (2006). Humic substances in soils Are they really chemically distinct Environ. Sci. Technol. 40,4605-4611, with the permission of the American Chemical Society.

Tseng, W.-L. Chiu, T.-C. Weng, J.-M. Chang, H.-T. Analysis of albumins using albumin blue 580, by capillary electrophoresis and laser-induced fluorescence. J. Liq. Chromatogr. Relat. Technol. 2001, 24 (19), 2971-2982. 

Albumin Blue 580 have been used to form stable and highly fluorescent complexes with proteins [59]. 

Adrenocmticotropin (ACTH), 221-222, 452, 462,496.498 Albumin Blue 574,565 Alcohol dehydrogenase. 333. 508 Alcohols, dielectric relaxadoa times. 225 Alkaline phosphatase, 259... 

Hofmann et al reported a first step toward a disposable diagnostic microchip for determination of urinary human semm albumin (HSA). They used an DEED based on a PPV derivative with a peak emission wavelength of 540 nm and which operated at drive voltages below 10 V. In a fluorescence assay, HSA was reacted with Albumin blue 580, generating a strong emission at 620 nm when excited with the OLED. Hie assay was performed in microchannels fabricated on a poly(drmethylsiloxane) microchip. HSA concentrations down to lOmgfr were detected, a limit suflicient for the determination of microalbuminuria, an increased urinary albumin excretion indicative of renal disease. 

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

The observed fluorescence blue shift following BLG adsorption was, however, small compared with the one observed for bovine serum albumin [28]. Univariate analysis... 

Jiang and Zhu (2000) and Qiu and Zhu (2001) have reported the fabrication of multilayered devices composed of stacks of compression-molded disks of alternating compositions. One type of disk is either P(SA-EG) or P[SA-co-TMAgly)-Z>-EG] and the other is a pH-sensitive, protein-loaded blend of, for example, poly(methacrylic acid) and polyethoxazoline. The release of model proteins, myoglobin, bovine serum albumin, and FITC-dextran, and compounds such as brilliant blue have been studied and pulsatile release profiles have been demonstrated (Jiang and Zhu, 2000 Qiu and Zhu, 2001). 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

Bromcresol green is frequently used for the quantitation of albumin, to which it binds selectively at pH 4.2 with a resulting increase in absorbance at 630 nm. The dye is initially a yellow colour but the resulting protein-dye complex is an intense blue colour (Figure 11.12). The method is relatively specific for albumin, bromcresol green being able to displace most substances that may be initially bound to the protein molecule. 

At 77 K the position of the 0-0 band is generally blue shifted for exposed tryptophans and red shifted for buried tryptophans. Along with a shift in wavelength to the red, the phosphorescence lifetime decreases.(28) The single tryptophan of human serum albumin shows red-shifted phosphorescence and D - L triplet zero-field splitting, indicating that it is in a hydrophobic environment. 29 ... 

Protein concentration was determined using the Bradford assay at 595 nm. 100 pL of the sample were introduced into a cuvette containing 5 mL of Bradford solution (100 mg of Coomassie blue, 50 mL of ethanol and 100 mL of 85 % phosphoric acid dissolved in 850 mL of H2O). The solutions were incubated for 5 min at room temperature. The absorbance was measured at 595 nm. The protein concentration in the sample was determined using a calibration curve plotted with serum albumin (1 mg mL ) as a standard.)... 

K. Murakami, T. Sano and T. Yasunaga, Bull. Chem. Soc. Japan 54, 862 (1981). This is unusual case where there are no temperature-jump relaxations. The interaction of bovine serum albumin with bromophenol blue is accompanied by four relaxations which are attributed to a fast second-order interaction followed by three first-order steps. 

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