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Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
Two techniques were routinely employed to detect the proteins separated by SDS-poly-aerylamide gel electrophoresis (PAGE) that is, silver staining and autoradiography of a gel of a radioiodinated-purified receptor. One faint band at Mr, ~32,000 was occasionally detected by Coomassie Brilliant Blue staining. However, silver staining detected at least nine major bands. Iodination of the affinity-purified receptor and analysis by SDS-PAGE also revealed several bands. When the iodinated receptor was repurified on a PRL-Affi-Gel column, only the Mr ~42,000 band was seen.50
Leptospira
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Tanu Sagar, Nitin Gupta, Rama Chaudhry
Microscopy: Direct demonstration of leptospires using dark ground microscopy can be done in the acute phase. The samples that can be used for diagnosis are blood, urine, and CSF. The problem that remains with direct demonstration is poor sensitivity and specificity [39]. For a leptospire to be visible, the load in sample should be as high as 104 leptospires/mL for 1 cell/field [40]. The sensitivity can be increased by double centrifugation of the sample. In double centrifugation, the first centrifugation is at low speed to separate the cellular elements, followed by high-speed centrifugation to concentrate leptospires. Also, direct demonstration has poor specificity, as leptospires can be mistaken with artefacts like lysed red blood cells and fibrils. Techniques such as silver staining, immunoperoxidase staining, and immunofluorescent staining can be used for tissue samples.
Analysis of Normal and Neoplastic Tissue NHC Proteins by High-Resolution Two-Dimensional Gradient Electrophoresis and Silver Staining
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Due to difficulty in obtaining sufficiently high specific activity of isotypically labeled proteins from tissues labeled in vivo, we utilize a method in which high specific activity protein labeling is accomplished in vitro. Display of radioactively labeled proteins serves as an adjunct to silver staining since it is not known whether silver stains or detects all types of proteins stoichiometrically. These two diverse methods allow for detection and quantitation of nearly all proteins of interest to us. Known amounts of lyophilized samples are reductively methylated with [14C] or [3H]-formaldehyde (New England Nuclear, specific activity 42 to 48 mCi/mmol) which has been purified by passage through a small Dowex® 1-X8 column. Reductive methylations are carried out in a 1-mℓ reaction volume containing 500 to 1000 μg of protein,96 2 mM [14C]-formaldehyde (100,000 dpm/(μmol), 20 mM NaCNBH3 in 100 mM Hepes® [4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid] buffer and 8 M urea. After incubation at room temperature (22 to 25°C) overnight, 3 mℓ of 10% trichloroacetic acid (TCA) is added. Precipitated samples are isolated by centrifugation, a portion aliquoted, dissolved in 0.2 mℓ of 0.2 M NaOH and counted in Scintiverse® (Fisher). In test methylations of bovine serum albumin (BSA) and whole rat heart homogenates, we routinely obtained specific activities approaching 80 to 150,000 dpm/μg of protein. Gels containing 14C or 3H labeled proteins are processed by scintillation autography (fluorography) using X-ray film (Figure 3).94 By co-electrophoresis of 14C and 3H in vitro labeled NHC proteins extracted from different test chromatins and a combination of fluorography and autoradiography,61,88 comparisons with high confidence in a single matrix are possible. In addition, recent tests indicate that gels containing 35S, 14C, or 3H labeled proteins may be, with low quenching effect, first stained with silver and then processed by autoradiography and fluorography.65,103
Development of a method of nasal secretions sampling for local nasal inflammation studies
Published in Expert Review of Clinical Immunology, 2023
Xu Xu, Xu Zhang, Dong Liu, Kunpeng Wang, Yang Wang, Chengshuo Wang, Yuan Zhang, Jingyun Li, Luo Zhang
To compare the proteins in nasal secretions collected by the two methods, we used silver staining. Silver staining is an excellent technique for detecting proteins separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and efficiently detects the presence of proteins in nanograms [15]. The staining process mainly includes: (1) fixation to get rid of interfering compounds; (2) sensitization and rinses to increase the sensitivity and contrast of the staining; (3) silver impregnation with either a silver nitrate solution or a silver-ammonia complex solution; (4) rinses and development to build up the silver metal image; (5) stop and rinse to end development prior to excessive background formation and to remove excess silver ion and other chemicals prior to further processing [16].
Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics
Published in Expert Review of Proteomics, 2022
(2) After the first-dimensional SDS-PAGE, the gel is usually stained with acetic acid/methanol solution containing CBB and destained with acetic acid/methanol solution. Proteins separated by first-dimensional SDS-PAGE are precipitated with acid/methanol solution to immobilize on the gel. It is essential to prevent the loss of proteins during the equilibration of the gel with the stacking gel buffer before the second-dimensional Phos-tag SDS-PAGE is performed and estimate the quantity of proteins and their relative molecular weights. Silver and fluorescent stainings can be conducted after SDS-PAGE. However, although CBB staining is less sensitive than silver and fluorescent stainings, it is easier to perform than silver staining and allows performing a more quantitative analysis than silver and fluorescent stainings. After CBB staining, the pH of the gel becomes low; therefore, it should be adjusted to the same pH as that of stacking gel buffer solution before performing Phos-tag SDS-PAGE in the second dimension.
Different isolation approaches lead to diverse glycosylated extracellular vesicle populations
Published in Journal of Extracellular Vesicles, 2019
Daniela Freitas, Meritxell Balmaña, Juliana Poças, Diana Campos, Hugo Osório, Andriana Konstantinidi, Sergey Y. Vakhrushev, Ana Magalhães, Celso A. Reis
EVs were successfully isolated from both MKN45 and gMKN45 cell lines using the four aforementioned methodologies. Silver staining was used for total protein profile analysis and as protein loading control (Figure 5). The total protein profile of the two cell lines was similar whenever the same methodology and culture condition were applied. However, the different EV isolation methodologies affected the protein profile obtained for both cell lines. Moreover, the presence or the absence of FBS in the culture medium also showed to impact the total protein profiles for both MKN45 and gMKN45 cell lines. In particular, TEI led to the largest co-isolation of BSA and ODG to the least (Figure 5). The band around 65 kDa was identified by MS as BSA (Figure 5). Some additional contaminant bovine proteins were detected in the EV samples isolated from the cells cultured with medium supplemented with FBS (Figure 5). In the EV samples collected from non-supplemented medium, common EV markers, such as syntenin-1 or CD9, were detected by MS (Figure 5), and then validated by Western blot in both culture conditions (Figure 6(a,b)).