Abstract Celia's Encephalopathy (MIM # 615924 ) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin ...(Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6–8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers.
A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from ...conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.
Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major ...constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu2+) to cuprous ions (Cu1+), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.
► Photopolymerization chemical bases are presented. ► Approaches to develop and amplify biorecognition events are discussed. ► Protein and DNA interaction events using the photopolymerization ...principle are shown. ► Nanomolar sensitivity for protein-to-protein (or antigen) recognition is achieved. ► Compact-disc technology is successfully applied to quantify PMR events.
This work addresses the main topics related to the photopolymerization process to develop and to monitor the analytical signal derived from biorecognition assays. We review basic aspects of photopolymerization, together with the nature and the reactivity of the chemicals involved. Focusing on DNA and protein determination and medical applications, we envisage as relevant photopolymers that generate biocompatible hydrogels.
We also review the major characterization techniques applied to ascertain the composition of the products formed and to monitor the progress of the reaction. In this sense, we present optical microscopy, fluorescence imaging, ATR-FTIR spectroscopy and profilometry measures.
Finally, we discuss analytical examples and applications of protein-to-protein, protein-to-antigen and DNA-hybridization photopolymerization. We demonstrate implementation of CD/DVD player technology to the detection and quantitation of such biointeractions, highlighting the advantages of this powerful methodology in biosensing.
CdTe quantum dots were synthesized in aqueous solution using 3-Mercaptopropionic acid as stabilizing agent. Chemically reduced ovalbumin had been used to modify the surface of CdTe QDs, resulting in ...the enhancement of photoluminescence intensity and the blue shift of the PL peak. A new method for the determination of ovalbumin is established based on the PL changes. Under the optimal conditions, experiment results showed that the PL intensity and the shift of PL peak are both in good linear with the ovalbumin concentration in the range of 0.125–4
μM and the detection limit was 0.03
μM. The method is compared with the traditional UV absorption method, and shows relatively good reliability.
A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of ...proteins with copper ions under strong alkaline conditions to form a copper–protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper–protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240
nm. Copper absorbance slowly increases from 240 to 190
nm where a blank with the reagents is recommended. A maximum absorbance for the copper–protein complex was found at 226
nm and high pH value. The stoichiometries of the copper–protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu
2+ ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu
2+ ions at pH 10.3. To determine proteins, 1–1.5
ml of 1.8% KOH solution with 0–20
μg
ml
−1 protein is treated with 25
mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226
nm in 1
cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1–20
μg
ml
−1 protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.
‘Ready-for-use’ instruments from surgical instrument trays were examined after routine cleaning and sterilization in a blinded study. These reprocessed instruments originated from five National ...Health Service hospital trust sterile service departments in England and Wales. Determination of residual protein and peptide contamination was carried out by acid stripping of the instrument surfaces, hydrolysis of the constituent amino acids and quantitative total amino acid analysis. One hundred and twenty instruments were analysed, and the median levels of residual protein contamination per instrument for the individual trays were 267, 260, 163, 456 and 756
μg. Scanning electron microscopy and energy dispersive X-ray spectroscopic analyses of the instruments showed that tissue deposits were localized on surfaces, but there was no significant correlation between overall protein soiling and instrument complexity. The highest levels of residual contamination were found on instruments used for tonsillectomy and adenoid surgery.
In the present work, the determination of the total protein concentration in hyperimmune serum samples was performed through a partial least-squares near-infrared (NIR-PLS) method. The method was ...based on the chemometric treatment of the NIR spectra of samples. The influences of spectra preprocessing and spectral window utilized in the construction of PLS model were studied. Models were built using reference data of 19 samples selected through the use of hierarchical cluster analysis (HCA) of NIR spectra of samples and another 24 samples were employed for external validation of the method. A model with better prediction capacity was obtained after whole spectra preprocessing by multiplicative scattering correction (MSC) algorithm and using data in the spectral range of 2158–2209nm. Under optimized conditions a RMSEP of 0.21gdl−1 and a quality coefficient value (QC) of only 5.8% were obtained for the prediction of total protein content in the samples used for external validation. Also, a determination coefficient, r2, of 0.97 was obtained in the correlation of predicted and reference data of samples situated in the validation set.
To analyze the reliability of determining the weight fraction of protein in the samples of pectic polysaccharides by the optical density of solutions in the ultraviolet and visible regions of the ...spectrum, a comparison of eleven methods: Flores, Lowry, Bradford, Sedmac, Ruhemann (ninhydrin reaction) methods, ultraviolet spectrophotometry, methods with Benedict reagent, with Nessler reagent, with amide black, with bicinchoninate reagent, and the biuret method has been carried out. As a result, the insufficient sensitivity of seven of the listed methods has been found, which does not recommend them for these purposes. The Lowry, Bradford, Sedmac methods, and the method with the Nessler reagent can be used for the determination of protein content in the samples of pectic polysaccharides, and the Bradford method can be recommended for characterization of samples of pectic polysaccharides if the weight fraction of protein does not exceed 15%, and the Lowry method can be used for the samples with the weight fraction of protein greater than 15%.
A simple, sensitive and selective method was proposed for the determination of proteins by using a resonance light scattering technique. The weak resonance light scattering (RLS) of Bordeaux red (BR) ...can be enhanced greatly in the pH range 3.87–3.96 by the addition of micro amounts of proteins, resulting in four characteristic peaks in the wavelength range 250–600
nm. At the maximal wavelength of 363
nm, the enhanced RLS is proportional to the concentration of proteins in the range 0.12–10.8
μg
ml
−1 for bovine serum albumin (BSA) and 0.24–18.0
μg
ml
−1 for human serum albumin (HSA). The detection limits were 40.0 and 52.9
ng
ml
−1 for BSA and HSA, respectively. The present method has been applied to the determination of total proteins in human serum, urine and saliva samples. The obtained results are in good agreement with those obtained by the Bradford method with relative standard deviations (R.S.D.) of 0.9–2.3%.
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