RAW264.7 cells (1 × 106 cells/well) were seeded into a 6-well tissue culture plate and cultured overnight. Pre-treatment and infection of cells were the same as in invasion assay. At 30 min pi, cell lysates were collected, protein concentrations were determined using Bradford protein assay and were boiled for 5 min in 2x Laemmli sample buffer (Bio-Rad Lab. Inc., USA). Immunoblot assay was performed with slight modifications as we previously described [11 (link)]. Briefly, membranes were incubated with phospo-specific antibodies against ERK, JNK and p38α (1:250, Cell Signaling Technology, Inc., USA) in 5% bovine serum albumin (GenDEPOT, USA) at 4°C overnight. Incubation with secondary antibody was done using peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, USA; 1:1,000 dilution) at room temperature for 1 h. β-actin antibody was used as a loading control. Membranes were exposed to a Molecular Imager ChemiDoc XRS+ system machine (Bio-Rad Lab.). NHI ImageJ software (USA) was used to quantify the immunoblots.
Molecular imager chemidoc xrs system machine
Manufactured by Bio-Rad
Sourced in United States
The Molecular Imager ChemiDoc XRS+ system is a laboratory imaging instrument designed for the detection and analysis of chemiluminescent, fluorescent, and colorimetric signals. The system utilizes a charge-coupled device (CCD) camera and specific optical filters to capture high-quality images of electrophoresis gels, Western blots, and other life science applications.
Automatically generated - may contain errors
2 protocols using molecular imager chemidoc xrs system machine
1
Western Blot Analysis of MAPK Signaling
2
Emodin Modulation of MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells were prepared and pre-incubated with emodin or PBS as in the flow cytometry for F-actin content determination.
After washing, the cells were incubated at 4°C overnight using ice-cold radioimmunoprecipitation assay (RIPA) buffer with 1% protease inhibitor cocktail. The cell lysates were collected and the protein concentration was measured using the Bradford protein assay (Bio-Rad, USA). Proteins were separated by SDS-PAGE and electrically transferred onto Immobilon-P membranes (Millipore, USA). The membranes were incubated with mitogen-activated protein kinases (MAPKs) (ERK1/2, JNK and p38α) as previously described [3] , exposed to a Molecular Imager ChemiDoc XRS+ system machine (Bio-Rad, USA) and the immunoblot signals were quantified using NIH ImageJ software. In addition, we determined the effect of ERK activator, MEK-1 (Santa Cruz Biotechnology, Inc., Germany) on the internalization of Brucella in emodin-incubated cells. The same procedures were performed as in the bacterial invasion assay wherein after pre-incubation of macrophages with emodin, the cells were incubated with MEK-1 (3 μg/well) for 45 min prior to infection.
After washing, the cells were incubated at 4°C overnight using ice-cold radioimmunoprecipitation assay (RIPA) buffer with 1% protease inhibitor cocktail. The cell lysates were collected and the protein concentration was measured using the Bradford protein assay (Bio-Rad, USA). Proteins were separated by SDS-PAGE and electrically transferred onto Immobilon-P membranes (Millipore, USA). The membranes were incubated with mitogen-activated protein kinases (MAPKs) (ERK1/2, JNK and p38α) as previously described [3] , exposed to a Molecular Imager ChemiDoc XRS+ system machine (Bio-Rad, USA) and the immunoblot signals were quantified using NIH ImageJ software. In addition, we determined the effect of ERK activator, MEK-1 (Santa Cruz Biotechnology, Inc., Germany) on the internalization of Brucella in emodin-incubated cells. The same procedures were performed as in the bacterial invasion assay wherein after pre-incubation of macrophages with emodin, the cells were incubated with MEK-1 (3 μg/well) for 45 min prior to infection.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!