Ab232401

Manufactured by Abcam

Sourced in United States

Ab232401 is a lab equipment product offered by Abcam. It is a piece of laboratory equipment designed for a specific scientific function. No further details about its core function or intended use are provided without the risk of an unbiased or extrapolated description.

Automatically generated - may contain errors

Lab products found in correlation

3 protocols using ab232401

Protein Expression Analysis by Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein was extracted using RIPA buffer (Solarbio Science & Technology, Beijing, China) with 1 % protease inhibitor, and concentration was determined with a BCA kit (Biosharp, Anhui, China) following the manufacturer's protocols. The protein sample (10 μg/lane) was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane. Then, the membranes were blocked with 5 % non-fat milk for 1 h, and then incubated with primary antibodies, including TLR4 (ab13556, 1:500 dilution, 96 kDa, Abcam), NLRP3 (ab232401, 1:500 dilution, 118 kDa, Abcam) and GAPDH (ab245355, 1:3000 dilution, Abcam) overnight at 4 °C. Then the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (ab205718, 1:8000 dilution, Abcam) for 2 h. Finally, the blots were visualized using ECL reagent (Meilun Biotech, Dalian, China). The grey values of proteins were analyzed utilizing Image J software (NIH, Bethesda, MD, USA). Relative protein expression was normalized to the control group, with GAPDH as a loading control.

Yang J., Wei Z., Li H., Lv S., Fu Y, & Xiao L. (2024). Paeoniflorin inhibits the inflammation of rheumatoid arthritis fibroblast-like synoviocytes by downregulating hsa_circ_009012. Heliyon, 10(9), e30555.

+ Open protocol

+ Expand

Coimmunoprecipitation of NLRP3 and USP14

Check if the same lab product or an alternative is used in the 5 most similar protocols

For exogenous coimmunoprecipitation experiments, AF cells were cotransfected with Flag-NLRP3 and/or HA-USP14 expression plasmids. At 24 h posttransfection, the cells were harvested and lysed with 500 μL of RIPA lysis buffer containing protease cocktail. Lysates were centrifuged at 14,000
g for 5 min. The supernatant was transferred to a new tube and precipitated with 20 μL of anti-Flag or anti-HA affinity gel (Biotool, Houston, USA) for 2 h at 4°C. For
in vivo coimmunoprecipitation, the extracted protein from AF cells was incubated with Protein A/G PLUS-Agarose (sc-2003; Santa Cruz Biotechnology, Santa Cruz, USA;) for 1 h and then with anti-USP14 antibody (ab137432; Abcam), anti-NLRP3 antibody (15101; Cell Signaling Technology), or normal IgG (sc-2027; Santa Cruz Biotechnology) overnight at 4°C. Then, protein A-sepharose was used to pull down the immunocomplexes and the immunocomplexes were analysed by western blot analysis using anti-USP14 (ab192618; Abcam), anti-NLRP3 (ab232401; Abcam) or anti-ubiquitin (ab7780; Abcam) antibodies.

Hai B., Mao T., Du C., Jia F., Liu Y., Song Q., Pan X., Liu X, & Zhu B. (2022). USP14 promotes pyroptosis of human annulus fibrosus cells derived from patients with intervertebral disc degeneration through deubiquitination of NLRP3: USP14 induces deubiquitination of NLRP3. Acta Biochimica et Biophysica Sinica, 54(11), 1720-1730.

+ Open protocol

+ Expand

Protein Extraction and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein was extracted using a RIPA lysis buffer including a protease inhibitor combination (Sigma-Aldrich) that had been freshly added. Supernatants were collected after centrifugation. Bicinchoninc acid reagent (Thermo Scientific) was applied to assess protein concentration. The cytoplasmic and nuclear levels of NF-κBp65 were examined using an NE-PER kit (Thermo Scientific). Sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Millipore, Bedford, USA) were used to isolate the proteins and then transferred onto nitrocellulose membranes. After blocking in 5% non-fat dry milk, the membranes were then incubated overnight with the following antibodies: TRIM64 (Novus Biologicals, LLC, Centennial, CO, USA; NBP2-83,713), CD36 (Abcam; ab133625), ABCA1 (Abcam; ab18180), NF-κB (Abcam; ab16502), NLRP3 (Abcam; ab232401), ASC (Abcam; ab155970), caspase-1 (Abcam; ab207802), GSDMD-N (Abcam; ab215203), anti-IκBα (Abcam; ab32518), H3 (Cell Signaling Technology; 4499), and GAPDH (Cell Signaling Technology; 5174) followed by the horseradish peroxidase-conjugated secondary antibody. Immunoreactive band was visualized with enhanced chemiluminescence detection kit reagents (Servicebio, Wuhan, China).

Zhu C., Chen W., Cui H., Huang Z., Ding R., Li N., Wang Q., Wu F., Zhao Y, & Cong X. (2022). TRIM64 promotes ox-LDL-induced foam cell formation, pyroptosis, and inflammation in THP-1-derived macrophages by activating a feedback loop with NF-κB via IκBα ubiquitination. Cell Biology and Toxicology, 39(3), 607-620.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!