China 
Africa 
Explore chapters and articles related to this topic
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
For protein isolation, samples stored at −80°C are first pulverised in liquid nitrogen (LN2) prior to homogenisation on ice, using handheld or machine homogenisers (described above) in relevant lysis buffer (depending on the analyses to be performed e.g. Western blotting and proteomics as described later in this chapter), typically contain protease and phosphatase inhibitors, that prevent proteins from degrading and halt any continued phosphorylation. Preparation on ice is also important (in combination with phosphatase inhibitors), particularly if phospho-protein analyses are to be performed. Following homogenisation, samples are centrifuged at, e.g., 1,000 g at 4 °C for 5 minutes. Soluble proteins are contained within the supernatant and non-soluble (e.g. myofibrillar proteins) in the ensuing pellet. The pellet can be resuspended in relevant assay buffer and protein concentrations determined by specific protein assays such as the bicinchoninic acid (BCA) or Bradford protein assay, prior to aliquoting for long-term storage or for further analyses. As an example, the BCA protein assay detects Cu+1, and is created when Cu+2 is reduced by protein in an alkaline environment (14). When the reagent supplied in the BCA assay is mixed with the protein sample, two molecules of BCA join with one cuprous (Cu+1) molecule and the reaction becomes purple-coloured. This purple colour is due to the macromolecular structure of protein, the number of protein bonds and the presence of the four amino acids: Cysteine, Cystine, Tryptophan and Tyrosine (15). The darker the colour, the more protein present. The absorbance of this complex is then measured using a spectrometer between 540 and 590 nM which is linear with increasing protein concentrations.
Co-exposure to ambient PM2.5 plus gaseous pollutants increases amyloid β1–42 accumulation in the hippocampus of male and female rats
Published in Toxin Reviews, 2021
Saeed Motesaddi Zarandi, Abbas Shahsavani, Fariba Khodagholi, Yadolah Fakhri
To measure Aβ1–42 concentration in hippocampus tissue of rats, hippocampus tissue was homogenized in phosphate buffered saline (PBS). Three times the volume of hippocampus tissue, PBS solution in microtube was added. Microtubes homogenized using micro smash homogenizer MS-100 model (Tomy Seiko Co., Ltd, Tokyo, Japan) with 3000 rpm (4 °C) for 2 min. Then a solution prepared was centrifuged (Eppendorf Centrifuge 5415 R) at 3360 g at 4 °C for 10 min. Quantification of the total protein via Bradford assay was performed and was calculated required volume of sample to obtain equal protein mass (60 µg) (Bradford 1976). Then, Aβ1–42 concentration in the samples was detected using ELISA method. Protein concentration was normalized by the Bradford protein assay. ELISA assay was performed with rat Aβ1–42 ELISA kit (EZHS42; Millipore, Germany) (MBS 2009; Illouz et al. 2017).
cRGD peptide-conjugated polyethylenimine-based lipid nanoparticle for intracellular delivery of siRNA in hepatocarcinoma therapy
Published in Drug Delivery, 2021
Shuang Yang, Dandan Wang, Xia Zhang, Yaojun Sun, Bin Zheng
HepG2 cells were plated on 6 well plates (1 × 105 cells/well) overnight. Then, the medium was removed and cells were treated with PEI-NP/S, PSH-NP/S, cRGD-NP/S, or cRGD-PSH-NP/S for 4 h at 37 °C. Cells were cultured for another 44 h with a fresh medium. RIPA buffer containing 2% PMSF and 1% protease inhibitor cocktail was added to lyse the cells for 10 min at 4 °C. The lysed solution was collected and centrifuged at 10,000 rpm for 5 min to obtain the total proteins. Bradford protein assay was used to determine the concentration of protein. Then 40 μg total proteins were separated by 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% bovine serum albumin for 3 h and immunoblotted against GAPDH or survivin antibody at 4 °C overnight. The membrane was then incubated with HRP-conjugated goat anti-rabbit IgG for 4 h at 4 °C. ECL detection kits (GE Healthcare Life science, Waukesha, WI, USA) were used for detecting chemiluminescence.
The protective effect of fasudil against acrylamide-induced cytotoxicity in PC12 cells
Published in Drug and Chemical Toxicology, 2020
Mostafa Kianfar, Alireza Nezami, Soghra Mehri, Hossein Hosseinzadeh, A. Wallace Hayes, Gholamreza Karimi
The proteins involved in the apoptotic pathway including Bax, Bcl-2, caspase 3, 8, and 9, were detected by Western blot analysis. Briefly, after treatment of cells with Fasudil and ACR, cells were collected and lysed in a lysis buffer containing 50 mM Tris-HCl (pH: 7.4), 2 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM sodium orthovanadate (Na3VO4), 10 mM b glycerophosphate, 0.2% W/V sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a complete protease inhibitor mixture. Protein concentration of each sample was determined using the Bradford protein assay. Equal amounts of total proteins were loaded and fractionated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature. Then, blots were incubated with primary antibodies: Bax, Bcl-2, caspase-3, caspase-8, and caspase-9 at 1:1000 dilutions for 2 h at room temperature. After that, the membranes were washed with TBST three times and incubated with the secondary antirabbit antibody that conjugated with Horseradish-peroxidase at 1:3000 dilutions for 1 h at room temperature. After repeated washes with TBST, membranes were reacted with enhanced chemiluminescence (ECL) and the intensity of bands was determined by Alliance 4.7 Geldoc (UK). Protein bands were analyzed using UVtec software (UK). Normalization of results was done against GAPDH intensities by mouse monoclonal antibody that is inactive against target proteins (Mehri et al. 2012, Razavi-Azarkhiavi et al. 2017; Makhdoumi et al. 2018).