Western blot analysis was performed to verify the expression level of methyl-accepting chemotaxis protein (McpC), a chemoreceptor in chemotaxis, of P. larvae after the treatment with N-MRJP2. The total amount of protein was extracted from cells, and the concentration was determined by Bradford Kit (Sangon Biotech, Shanghai, China). Equal amounts of proteins were then separated by stacking (4%) and separating (12%) SDS-PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Invitrogen). After being blocked with 5% skimmed milk in Tris-buffered saline with Tween (TBST) 20 for 1 h, the membrane was washed and incubated with a primary antibody of McpC (Signalway antibody, ShenYang, China) at a dilution of 1:2000, then washed with TBST three times and incubated with secondary antibodies (goat anti-mouse, Invitrogen) at room temperature for 1 h. The blots were visualized using the Chemic Doc XRS imaging system (Bio-Rad) in the presence of enhanced chemiluminescence (Pierce, Thermo Fisher). The targeted protein band was quantified by NIH Image J software and normalized by Sodium Potassium ATPase, which was used as a loading control and its antibody was purchased from Abcam (Cambridge, MA, USA).
Chemic doc xrs imaging system
Manufactured by Bio-Rad
Sourced in China
The Chemic Doc XRS imaging system is a versatile laboratory equipment used for capturing and analyzing images of chemiluminescent, fluorescent, and colorimetric samples. It provides high-resolution imaging capabilities for a variety of applications in life science research and analysis.
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Lab products found in correlation
2 protocols using chemic doc xrs imaging system
1
Quantifying Bacterial Chemoreceptor Expression
2
Western Blot Analysis of α-SMA
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To investigate the expression level of α-SMA, a VSMC-specific marker, after being treated with MRJP1, Western blot analysis was also performed following the standard procedure. The total amount of protein was extracted from cells, and the concentration was determined by Bradford Kit (Sangon Biotech, Shanghai, China). Then equal amounts of proteins were separated by stacking (4%) and separating (12%) SDS-PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Invitrogen). After being blocked with 5% nonfat milk in Tris buffered saline with Tween 20 (TBST) at 37 °C for 1 h, the membrane was washed and incubated with primary mice antibodies for α-SMA at a dilution of 1:500. Then the membrane was washed with TBST three times and incubated with secondary antibodies (goat antimouse, Invitrogen) at room temperature for 1 h. The blots were visualized using the Chemic Doc XRS imaging system (Bio-Rad) under the presence of ECL Western blotting substrate (Pierce). The bands were quantified using Quantity One software (v4.6.2, Bio-Rad) and normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as a loading control.
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