Fructose-1,6-bisphosphate aldolase (FBA) of MS was previously identified as a cytoplasmic protein [56 ] and was used as a cytoplasmic protein control in this research. MSnox and MSfba gene fragments were amplified from MS WVU1853 by overlap PCR with the primers shown in Table S1 , which have been described in our previous studies [26 , 56 ]. The MSnox and MSfba fragments were ligated into pET28a ( +) (Novagen, USA), and the recombinant strain E. coli BL21 (pET28a-MSnox) and E. coli BL21 (pET28a-MSfba) were constr0-ucted. Then the His-tagged rMSNOX protein and rMSFBA protein were expressed and purified as described previously [26 , 56 ]. The purified rMSNOX protein and rMSFBA protein were analyzed with 12.5% SDS-PAGE, stained with Coomassie blue-G250 (Solarbio, China), and imaged with an infrared laser scanning imaging system (Ddyssey; LI-COR, USA). The protein concentrations were detected with a BCA protein assay kit (Beyotime, China).
Coomassie blue g250
Manufactured by Solarbio
Sourced in China
Coomassie blue-G250 is a dye used in biochemical assays for the quantitative determination of protein concentrations. It binds to proteins and produces a blue color that can be measured spectrophotometrically. The intensity of the blue color is directly proportional to the amount of protein present in the sample.
Automatically generated - may contain errors
3 protocols using coomassie blue g250
1
Purification and Characterization of MS NADH Oxidase and Aldolase
2
Characterization of Elaeagnus mollis Oil Meal
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Elaeagnus mollis oil (EMO) meal, a by-product of EMO extraction by utilizing supercritical carbon dioxide extraction technology, was provided by Shanxi Qierkang Samara Biological Products Co., Ltd. (Taiyuan, China). The EMO meal had 5.27 g water/100 g, 49.43 g protein/100 g, 22.3 g fat/100 g, and 3.32 g ash/100 g sample. Bovine serum albumin (BSA), alcalase (200,000 IU/g) and Coomassie blue G-250 were purchased from Beijing Solarbio Science &Technology Co., Ltd. (Beijing, China). Tris (hydroxymethyl) aminomethane (Tris), β-mercaptoethanol (β-Me), sodium dodecyl sulfate (SDS) and 8-aniline-1-naphthalenesulfonic acid (ANS) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the chemicals used in this experiment were of analytical grade.
3
Acrosome Reaction Triggered by Calcium Ionophore and Progesterone
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Following a 60-min period of in vitro capacitation, the spermatozoa underwent extended incubation for an extra 30 min within the same medium, supplemented either with 10 μmol l−1 A23187 calcium ionophore or 10 μmol l−1 progesterone (Sigma-Aldrich, St. Louis, MO, USA) to trigger the acrosome reaction. In contrast, the control samples were exclusively exposed to the dimethyl sulfoxide (DMSO) solvent. After treatment, the sperm were subjected to fixation and stained using Coomassie Blue G-250 (Solarbio, Beijing, China) in accordance with previous methodologies.28 (link)29 (link) At least 200 cells were counted for every sample, wherein the evaluation focused on determining the occurrence or absence of deep blue staining within the acrosomal crescent situated on the anterior facet of the sperm head. Subsequently, the proportions of acrosome reactions triggered by A23187 and progesterone (Sigma-Aldrich, St. Louis, MO, USA) were calculated, adjusting for the baseline levels noted in the DMSO controls.
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