The determination of salt-soluble protein was based on Wu et al. (2014) (link) with minor modification. One-gram prawn muscle was added with 15 ml cooled ultrapure water and then homogenized (F6/10, FLUKO, Germany) for 30 s on ice. The homogenate was extracted at 4 °C for 20 min and centrifuged at 10,000 g using a centrifuge at 4 °C for 20 min (CF16RXII, HITACHI, Japan). Supernatant was removed, and precipitate was added with 15 ml Tris-maleate buffer (0.6 M NaCl-20 mM Tris-maleate, pH 7.0) and then homogenized for 30 s. Then, the homogenate was kept at 4 °C for 60 min to extract salt-soluble protein and centrifuged at 10,000 g at 4 °C for 20 min. The obtained supernatant was diluted to 25 ml (0.6 M NaCl-20 mM Tris-maleate, pH 7.0), which was myofibrillar protein solution for quantitative determination. Salt-soluble protein content was determined using a kit according to the Bradford method (Solarbio, China). For each treatment, three replications were performed.
Bradford method
Manufactured by Solarbio
Sourced in China
The Bradford method is a spectrophotometric analytical procedure used to measure the concentration of protein in a solution. It is a colorimetric assay that relies on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured at a specific wavelength. The Bradford method provides a quick and reliable way to quantify protein levels in various samples.
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Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Cf16rxii, by Hitachi (1 mentions)
Hmga2, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Proliferating cell nuclear antigen (pcna), by Wanlei (1 mentions)
Gapdh, by Wanlei (1 mentions)
Itraq reagent, by Thermo Fisher Scientific (1 mentions)
Strata x c18 column, by Phenomenex (1 mentions)
Ultremex scx column, by Phenomenex (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
β tubulin, by Proteintech (1 mentions)
P erk1 2, by Wanlei (1 mentions)
Ccnd2, by Proteintech (1 mentions)
Ki 67, by Wanlei (1 mentions)
α sma, by Wanlei (1 mentions)
Gapdh, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
P jnk, by Wanlei (1 mentions)
5 protocols using bradford method
1
Determination of Prawn Muscle Salt-Soluble Protein
2
Protein Expression Analysis in Aortic Smooth Muscle Cells
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Human aortic smooth muscle cells after siRNA‐treated for 48 hours were harvested, and total protein was extracted using RIPA buffer (Solarbio) and 1 mM PMSF (Solarbio). Protein concentrations were determined using the Bradford method (Solarbio). Equal amounts of protein samples were separated by SDS‐PAGE and transferred onto a PVDF membrane (Merck) after electroblotting. The membrane was blocked with 5% non‐fat milk in PBS for 2 hours and then was incubated with the primary antibodies overnight at 4°C: HMGA2 (1:1000, Proteintech), PCNA (1:1,000, Wanleibio), SM22α (1:1,000, CUSABIO) and GAPDH (1:1,000, Wanleibio). After three 5‐minutes washes with TBS‐tween, the membrane was then incubated with goat anti‐rabbit IgG secondary antibody or rat antimouse secondary antibody (dilution at a 1:2000, Wanleibio) at 37°C for 2 hours. The membranes were visualized by ChemiDoc™ MP Imaging System (BIO‐RAD).
3
Pooled Sample Preparation and Proteomic Analysis
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To reduce individual differences, a pooled sample was prepared from the mixture of 15 samples (with the equal amount) in groups EH and TJT, respectively. Thus, there were 4 pooled samples (Groups EH1, EH2, TJT1, and TJT2) which would be analyzed in following experiment. The pooled samples were reduced with DTT (10 mmol/L) and alkylated with IAM (55 mmol/L). Then, samples were precipitated by cold acetone at −20°C overnight. After centrifugation at 30,000g under 4°C, the pellet was dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging, total protein concentration of the supernatant was measured with Bradford method (Solarbio, Beijing, China) [13 (link)]. Proteins (with the amount of 100 μg) of each sample were digested with trypsin and labeled with iTRAQ reagents (Applied Biosystems, USA). EH1 and EH2 were labeled with iTRAQ 113 and 115 reagent, respectively; TJT1 and TJT2 were each labeled with iTRAQ 114 and 116 reagent.
The labeled samples were resolved into 20 fractions with an Ultremex SCX column containing 5 μm particles (Phenomenex, USA). The eluted fractions were then desalted with a Strata X C18 column (Phenomenex, USA) and dried under vacuum [14 (link)].
The labeled samples were resolved into 20 fractions with an Ultremex SCX column containing 5 μm particles (Phenomenex, USA). The eluted fractions were then desalted with a Strata X C18 column (Phenomenex, USA) and dried under vacuum [14 (link)].
4
Bioassay of Cry1Ac Toxin against P. xylostella
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The protoxin Cry1Ac used in this experiment was produced by Btk strain HD-73. Specific purification methods refer to the previous study [72 (link)], and the concentration of Cry1Ac was redetermined by the Bradford method (Solarbio, Beijing, China) before each use of the toxin. An artificial diet overlay assay [31 (link)] was used to determine the susceptibility of both mutant and wildtype strains of P. xylostella. Bioassays were performed on five replicates for seven different concentrations of the Cry1Ac toxin, with an insecticide-free control.
5
Western Blot Analysis of Protein Expression
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Proteins were extracted using RIPA buffer (Solarbio, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride (Solarbio). Protein concentration was determined using the Bradford method (Solarbio). Equal amounts of proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Merck, Germany). After blocking with 5% non-fat milk in TBST for 2 h, the membranes were incubated with primary antibodies against CCND2 (1:500, Proteintech, Wuhan, China), PCNA (1:1,000, Proteintech), Ki-67 (1:500, Wanleibio, Shenyang, China), p-ERK1/2 (1:300, Wanleibio), p-p38 (1:750, Wanleibio), p-JNK (1:300, Wanleibio), MMP9 (1:1,000, Wanleibio), MMP2 (1:500, Wanleibio), α-SMA (1:500, Wanleibio), GAPDH (1:1,000, Proteintech) and β-tubulin (1:1,000, Proteintech) at 4°C overnight. After washing with TBST, the membranes were then incubated with goat anti-rabbit IgG secondary antibody (dilution at a 1:20,000, Wanleibio) at 37°C for 2 h. Signal was visualized by ChemiDocTM MP Imaging System (BIO-RAD, California, USA). The experiment was repeated at least three times.
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