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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
A method known as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is then used to separate the denatured and negatively charged proteins within each sample by passing the samples through a gel constructed from acrylamide polymers, which cross-link to form a molecular sieve. Samples containing the standardised protein concentrations are loaded into wells at the top of the gel and an electric current is passed through the gel. This causes the negatively charged proteins to migrate towards the positive electrode at the bottom of the gel. Small proteins pass through the gel faster than large proteins and thus the procedure effectively orders proteins according to their size, i.e. their molecular weight in kDa (Figure 2.9).
Virus-Based Nanocarriers for Targeted Drug Delivery
Published in Devarajan Thangadurai, Saher Islam, Charles Oluwaseun Adetunji, Viral and Antiviral Nanomaterials, 2022
Semra Akgönüllü, Monireh Bakhshpour, Yeşeren Saylan, Adil Denizli
Virus-based nanocarriers have also been employed for other noninvasive imaging techniques, including PET (positron emission tomography). The principle of PET is based on the emission of radiation from a patient, radiopharmaceutical injected, is registered via external detectors positioned at several orientations (Omami et al. 2014). This technology frequently is used for longitudinal follow-up and cancer diagnosis. There are many platforms for use in the PET, such as mammalian viruses, bacteriophages, etc. (Shukla and Steinmetz 2015). For example; Farkas et al. modulated the nanoparticle-based agents, which are PEG chains addition. They labeled MS2 and MS2-PEG capsids that have DOTA chelators and injected them into mice that have tumour xenografts. As shown in Figure 9.7a, they used each MS2 coat protein monomer (amino phenylalanine (T19paF) and cysteine mutations) for facing exterior and interior surfaces. They attached maleimide-DOTA to the cysteines to let 64Cu binding for radio labeling (Figure 9.7b). They analysed disassembled proteins with an upper limit of conversion and attached PEG chains applying a fast reaction. They performed SDS-PAGE analysis in a short time to indicate 76% of the proteins (Figures 9.7c-e). They also compared with other capsids and observed that the MS2 agents depicted longer circulation times. Furthermore, the MS2 and MS2-PEG bacteriophages behaved the same, although the latter agent has remarkably less uptake in the spleen (Farkas et al. 2013).
The Chemistry of O-Polysaccharide Chains in Bacterial Lipopolysaccharides
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Acinetobacter, a species now being recognized as an etiological agent for nosocomial infections, has been divided into some 18 different species from DNA-DNA hybridization studies. Seven of these groups are named: A. calcoaceticus (DNA group 1), A. baumannii (DNA group 2), A. haemolyticus (DNA group 4), A. junii (DNA group 5), A. johnsonii (DNA group 7), A. Iwoffii (DNA group 8), and A. radioresistens (DNA group 12). O-serotyping schemes have also been devised with up to 34 distinct groups. The O-polysaccharides have conventional structures, and amino sugars are abundant (Table 9). Acinetobacter often occurs as R-forms, containing the complete or partial core but no O-antigen, but also with O-chains, sometimes with unusual features. One unusual feature is that for many species, the silver stain does not work in the SDS-PAGE analysis. Some species seem to contain more than one polymer. Thus, the A. baumanni 016 polymer may be a minor component in the O il strain. Pyruvic acid, an unusual component in LPS, is present in DNA group 1.
The impact of forced degradation conditions on mAb dimer formation and subsequent influence on aggregation propensity
Published in mAbs, 2022
Michael J. Knight, Léontine Floret, Nisha Patel, John O’Hara, Elizabeth Rodriguez
The isolated dimers were analyzed by SDS-PAGE (Figure 1c and Table 1). We noted some differences between the apparent molecular weight observed by SDS-PAGE and the theoretical molecular weights for mAb1 (LC ~ 25 kDa, HC ~ 50 kDa, Monomer ~ 150 kDa). Migration on an SDS-PAGE gel can be influenced by many other factors than molecular weight, such as the pI of the molecule, folding state, and amount of SDS bound. In addition, the use of pre-stained molecular weight standards can result in discrepancies between apparent and theoretical molecular weight.34 Despite this, given the highly purified nature of these samples it was possible to assign the species present based on their relative migration. For all three dimer samples, analysis under non-reduced conditions revealed the presence of SDS-resistant dimer, the majority of which reduce to heavy chain and light chain when analyzed under reducing conditions. This suggests that a proportion of the dimer species in all three samples may be in part held together via disulfide bonds. This is in good agreement with previous work which has shown a significant contribution from disulfide bonds to mAb dimer formation.4
Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics
Published in Expert Review of Proteomics, 2022
Kinoshita et al. [14,18,19] and Kinoshita-Kikuta et al. [12,15–17] developed Phos-tag gel electrophoresis using Zn2+ instead of Mn2+. When performing SDS-PAGE using Mn2+-Phos-tag under alkaline pH conditions, gel stability did not last long and some phosphorylated proteins could not be detected. Thus, a method for separating phosphoproteins was developed that used a stable gel containing zinc (II) complex (Zn2+-Phos-tag acrylamide) at neutral pH. In this electrophoresis method, 357 mM N, N’-methylenebis (acrylamide) (BIS)-Tris-HCl buffer (pH 6.8) was used for separation and stacking gels and 0.10 M 3-morpholinopropanesulfonic acid (pH 7.8) containing 0.1 M Tris, 0.1% SDS, and 5.0 mM sodium bisulfite was used as the electrode solution. It is different from the discontinuous buffer system used by Laemmli [25]. Nonetheless, Kinoshita et al. [14,18,19] and Kinoshita-Kikuta et al. [12,15–17] succeeded in separating phosphoproteins that were difficult to separate by the Mn2+-Phos-tag system. Some researchers are interested in evaluating whether this method exhibits better resolution than SDS-PAGE that uses Laemmli discontinuous buffer system.
Intercellular transfer of mitochondria via tunneling nanotubes protects against cobalt nanoparticle-induced neurotoxicity and mitochondrial damage
Published in Nanotoxicology, 2021
Fuli Zheng, Zhousong Luo, Xinpei Lin, Wei Wang, Michael Aschner, Ping Cai, Yuan-Liang Wang, Wenya Shao, Guangxia Yu, Zhenkun Guo, Siying Wu, Huangyuan Li
Western blotting assay was conducted according to previous protocols (Zheng et al. 2019). Briefly, approx. 5 × 106 exposed cells were washed three times with cold PBS and collected by scraping. Collected cells were then lysed with 120 µL ice-cold RIPA lyse buffer (Beyotime, China). Afterwards, cell-free supernatants were obtained by centrifugation of the lysates at 12 000 g for 25 min at 4 °C. Sodium dodecyl sulfate (SDS) loading buffer was added to each supernatant, and subsequently boiled for 10 min to generate SDS-PAGE samples. 20 µL of samples were electrophoresed on a 10% SDS polyacrylamide gel. Proteins were then transferred onto a PVDF membrane. After blocking of the membrane with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at 25 °C, the blots were incubated with primary antibodies of interest overnight at 4 °C. After washing with TBST for 5 times, the blots were incubated with peroxidase-conjugated secondary antibody. Binding of antibodies was detected by chemiluminescence staining using the ECL detection kit (Amersham). The grayscale of protein bands was analyzed by Image J software. Primary antibodies were used at the following concentrations: HIF-1α (1:2000), β-ACTIN (1:10000), anti-rabbit and -mice peroxidase-conjugated secondary antibodies (1:10000).