For Immunoblot, whole cell lysate was obtained with low-salt lysis buffer after virus infection for indicated time points. Protein samples were mixed with the 5X loading buffer (Cell Signaling Technology) and resolved by SDS-PAGE. After electrophoresis, protein was transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories) and then incubated with appropriate antibody. LumiGlo Chemiluminescent Substrate System (KPL) was used to detect specific band of certain protein. Antibodies used in immunoblot were listed as follows. Anti-IRF3 rabbit polyclonal antibody, goat anti–mouse IgG-HRP and goat anti–rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-phospho-IRF3 (Ser396) rabbit monoclonal antibody, anti-RIG-I rabbit monoclonal antibody, anti-p38 MAPK rabbit polyclonal antibody, anti-phospho-p38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody, anti-SAPK/JNK rabbit polyclonal antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185) rabbit polyclonal antibody, anti-TBK1/NAK (D1B4) rabbit monoclonal antibody, anti-phospho-TBK1/NAK (Ser172) rabbit monoclonal antibody, IκBα muse monoclonal antibody were purchased from Cell Signaling Technology. Anti-IRF1 mouse polyclonal antibody was bought from abcam®.
Loading buffer
Manufactured by Cell Signaling Technology
Sourced in United States
5X loading buffer is a concentrated solution used to prepare samples for gel electrophoresis. It contains components that help maintain the sample's characteristics, such as density and pH, during loading into the gel. The buffer is designed to be diluted to a working concentration before use.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Anti irf3 rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit polyclonal anti p38 mapk antibody, by Cell Signaling Technology (1 mentions)
Anti phospho sapk jnk thr183 tyr185 rabbit polyclonal antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Polyvinylidene di uoride membrane, by Merck Group (1 mentions)
Primary antibody of runx 2, by Abcam (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
2 protocols using loading buffer
1
Immunoblot Analysis of Virus-Induced Signaling
2
Western Blot Protein Extraction and Analysis
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Radioimmunoprecipitation assay lysis buffer (Fude, China) was used to extract proteins from cells. Protein concentrations were measured using a BCA protein assay kit (Abcam, USA), according to manufacturer's protocols. Each sample (20 μg) was mixed with 5x loading buffer (Cell Signaling Technology, USA), and boiled for 10 min. After separation using SDS-PAGE and 4%-20% gradient gels, the proteins were transferred to 0.22 μm polyvinylidene di uoride membranes (Millipore, USA). The nonspeci c binding sites were blocked with 5% (wt/vol) skim milk for 120 min. The membranes were incubated with the primary antibody of RUNX2 (1:1000, abcam, USA) at 4 ℃ overnight. Subsequently, the membranes were incubated with horseradish peroxidase A niPure goat anti-mouse IgG secondary antibody (Emarbio, Beijing, China) at room temperature for 1 h. An enhanced chemiluminescence kit (Millipore Corp, Bedford, MA) was used for imaging.
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