Staining acidic phosphoproteins (phosvitin) in electrophoretic gels

Hegenauer, Jack; Ripley, Larry; Nace, George W.

doi:10.1016/0003-2697(77)90038-0

Cited by 81 publications

(41 citation statements)

References 11 publications

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“…An alternate method for visualizing phosphoproteins is based on trivalent metal chelation [220]. In this method, aluminum ions are added to an acidic CBB stain solution.…”

Section: Phosphorylated Proteinsmentioning

confidence: 99%

“…This promotes the formation of metal-protein chelates in which the aluminum reduces the negative charge and acts as a bridge between the dye and phosphate residue. This method could detect as little as 40 ng of apo-phosvitin, equivalent to 0.13 nmol of phosphate [220]. Like glycoproteins, a fluorescent dye for phosphoprotein detection is also now available.…”

Section: Phosphorylated Proteinsmentioning

confidence: 99%

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Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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“…An alternate method for visualizing phosphoproteins is based on trivalent metal chelation [220]. In this method, aluminum ions are added to an acidic CBB stain solution.…”

Section: Phosphorylated Proteinsmentioning

confidence: 99%

Section: Phosphorylated Proteinsmentioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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“…After dialysis against distilled water, the protein was lyophilized. Phosphate was detected by staining with Coomassie Brilliant Blue and aluminum nitrate (13). Carbohydrate was detected by the phenol-sulfuric acid method, periodic acid Schiff staining, or staining with a GelCode glycoprotein staining kit (Pierce).…”

Section: Methodsmentioning

confidence: 99%

“…However, aspolins are not likely to act as TMAO catalytic enzymes in vivo for several reasons. Because the circular dichroism spectrum of purified TMAOase at the neutral condition suggested the random coil structure (13), it is unlikely that TMAOase has a single effective active center. The low affinity of aspolin1/TMAOase for ferrous ion suggests that catalytic activity would be very low in vivo because the concentration of free ferrous ion in live cells is kept at a very low level, 0.2-0.5 M (28).…”

Section: Figmentioning

confidence: 99%

Aspolin, a Novel Extremely Aspartic Acid-rich Protein in Fish Muscle, Promotes Iron-mediated Demethylation of Trimethylamine-N-oxide

Takeuchi

Hatanaka

Kimura

et al. 2003

Journal of Biological Chemistry

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Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acidrich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala 42 and Asp 43 . Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5 part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca 2؉ -binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe 2؉ , whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe 2؉ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe 2؉ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat. Trimethylamine-N-oxide (TMAO)1 is widely distributed among various tissues of sea fish and invertebrates (1-3). Several physiological roles of TMAO in teleost have been proposed, i.e. osmoregulation, cryoprotectant, protection of proteins from high pressure, and disulfide formation (3). Gadoid fishes, including cod, pollack, hake, whiting, and haddock, are commercially important and contain large amounts of TMAO (30 -130 mmol/kg) in their muscle (1-3). In such species, TMAO is demethylated to dimethylamine (DMA) and formaldehyde during frozen storage (4, 5).(CH 3 ) 3 NO 3 (CH 3 ) 2 NH ϩ HCOH (Eq. 1)

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“…Phosvitin was stained under our conditions. It has been reported that this protein is not stained by Coomassie Blue (Hegenauer et al, 1977), unless trivalent metal ions are present, which were presumably provided by iron electrodes used during electrolytic destaining.…”

Section: Methodsmentioning

confidence: 99%

Proteins and lipoproteins in yolk from eggs of the estuarine crocodile (Crocodylus porosus); A comparison with egg yolk of the hen (Gallus domesticus)

Burley

Back

Wellington

et al. 1988

Comparative Biochemistry and Physiology Part B: Comparative Bio

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Abstract-1. The livetin patterns of the two species Crocodylus porosus and Gallus domesticus were very different and it was difficult to find a correspondence.2. In C. porosus the granules were a larger proportion of the total yolk but they contained very little lowdensity lipoprotein when compared with granules from hens' eggs.3. The low-density lipoprotein was a lower proportion of the yolk of C. porosus: 30% of the dry weight compared with 60% for the hen. Furthermore, the former apparently contained an unstable fraction consisting of large particles.4. The apoprotein patterns of the low-density lipoprotein of C. porosus eggs had a higher proportion of high-molecular weight constituents than that of the hen.5. A glycoprotein with M, approximately 2 x 10 4 has been isolated that does not correspond to a known avian protein.6. The characteristic protein of avian egg yolk, apovitellenin I, if present, was a minor constituent.

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Staining acidic phosphoproteins (phosvitin) in electrophoretic gels

Cited by 81 publications

References 11 publications

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Aspolin, a Novel Extremely Aspartic Acid-rich Protein in Fish Muscle, Promotes Iron-mediated Demethylation of Trimethylamine-N-oxide

Proteins and lipoproteins in yolk from eggs of the estuarine crocodile (Crocodylus porosus); A comparison with egg yolk of the hen (Gallus domesticus)

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