Protein isolation was performed simultaneously with RNA extraction from the cells. Briefly, 30 µg of extracted protein was loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Biorad, Germany), and blocked with 5% (w/v) non-fat dried milk (Merck, Germany) in PBS. The membrane was labeled with the rabbit anti-NF-κB p65 antibody (1:500, Abcam) or anti-GAPDH (Dako Cytomation, Denmark). The secondary anti body was goat anti-rabbit IgG-HRP (1:16000, Santa Cruz, USA). In case of the Flag antibody, HRPconjugated mouse anti-Flag antibody was used. Immunoreactive bands were visualized using an Amersham enhanced chemiluminescence (ECL) Advance Western Blotting Detection Kit (GE Healthcare, Germany). Western blots were carried out in triplicate from three separate cultures. The band intensity of NF-κB and p65 was compared that of GAPDH.
Rabbit anti nf κb p65 antibody
Manufactured by Abcam
Sourced in Denmark
Rabbit anti-NF-κB p65 antibody is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor. It can be used for the detection and analysis of NF-κB p65 in various applications such as Western blotting, immunohistochemistry, and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
Non fat dried milk, by Merck Group (1 mentions)
Anti gapdh, by Agilent Technologies (1 mentions)
Ecl advance western blotting detection kit, by GE Healthcare (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Rabbit anti eea1 antibody, by Cell Signaling Technology (1 mentions)
2 protocols using rabbit anti nf κb p65 antibody
1
Western Blot Analysis of NF-κB p65
2
Uptake and Localization of EVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells grown on sterile coverslips were incubated with TSG101-mCherry/CD63-EGFP-labeled EVs at 40% confluency. After 20–30 min, cells were washed extensively with PBS and then fixed with 4% paraformaldehyde for 20 min at room temperature followed by permeabilization with 0.3% Triton X-100 in PBS for 30 min. The samples were incubated with a blocking buffer containing 10% goat serum (NGS) in PBS for 2 hr at room temperature followed by addition of rabbit anti-Iba1 antibody (1:1,000; Wako Laboratory Chemicals) or rabbit anti-NF-κB p65 antibody (1:1,000; Abcam) or rabbit anti-EEA1 antibody (1:200; Cell Signaling Technology) and incubated overnight at 4°C. Primary Abs were labeled with secondary anti-rabbit Abs conjugated to the fluorescent probes Alexa Fluor 488 or Alexa Fluor 594, and nuclei were labeled with DAPI.
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