Ab205236

Manufactured by Abcam

Sourced in United States

Ab205236 is a lab equipment product from Abcam. It is designed for use in research applications. The core function of this product is to provide a tool for researchers to conduct their experiments.

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Lab products found in correlation

5 protocols using ab205236

Western Blot Analysis of Inflammatory Markers

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Cells and tissue samples were lysed for protein extraction using RIPA assay. The concentration of each protein sample was determined by a BCA (bicinchoninic acid) kit. Equal amounts (50 mg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, United States). Membranes were blocked using 5% non-fat dry milk with TBST buffer for 1 h, then incubated overnight at 4° C with GPR43 (1:1000, ab131003, Abcam), MFN2 (1:500, ab205236, Abcam), PPARγ (1:1000, ab178860, Abcam), NOX-1 (1:1000, ab121009, Abcam), EBP50 (1:1000, ab3452, Abcam), p47phox (1:1000, ab166930, Abcam), NLRP3 (1:1000, 15101, Cell Signaling Technology, Danvers, MA, US), Caspase-1 (1:1000, sc-392736, Santa Cruz Biotechnology), IL-1β (1:1000, 12242, Cell Signaling Technology, Danvers, MA, US), and β-Actin (1:5000, sc-47778, Santa Cruz Biotechnology). After washing, membranes were probed further with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, Santa Cruz Biotechnology). After washing with TBST for 15 min, immunoreactive bands were exposed by enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).

Zhang W., Wang W., Xu M., Xie H, & Pu Z. (2021). GPR43 regulation of mitochondrial damage to alleviate inflammatory reaction in sepsis. Aging (Albany NY), 13(18), 22588-22610.

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Quantifying Mitochondrial Dynamics Proteins

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Total protein was extracted from cells with RIPA lysis buffer (R0010, Solarbio, Beijing, China). A bicinchoninic acid (BCA) protein assay kit (P0012, Beyotime, Shanghai, China) was used to determine the protein concentration. The protein was detected by sodium dodecyl sulfat-polyacrylamide gel electrophoresis and electrically imprinted on the membrane. Membranes were incubated with primary antibodies PGC1-α (1:1000, ab106814, Abcam, UK), LC3 (1:1000, ab63817, Abcam, UK), Beclin 1 (1:2000, ab207612, Abcam, UK), PINK1 (1:1000, ab300623, Abcam, UK), Parkin (1:2000, ab77924, Abcam, UK), Mfn1 (1:1000, ab221661, Abcam, UK), Mfn2 (1:2000, ab205236, Abcam, UK), OPA1 (1:2000, ab42364, Abcam, UK), GAPDH (1:2000, ab8245, Abcam, UK) at 4℃ overnight, then incubated with horseradish peroxidase (HRP) labeled secondary antibody (1:2000, ab97051, Abcam, UK). Enhanced chemiluminescence (ECL) kit (WP20005, Thermo Fisher Scientific, Waltham, USA) was used for color development. Finally, the gray values of protein bands were analyzed by ImageJ software.

Mu N., Zhang T., Zhu Y., Lu B., Zheng Q, & Duan J. (2023). The mechanism by which miR-494-3p regulates PGC1-α-mediated inhibition of mitophagy in cardiomyocytes and alleviation of myocardial ischemia—reperfusion injury. BMC Cardiovascular Disorders, 23, 204.

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Mitochondrial Dynamics Protein Expression

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Protein expression was measured using western blotting. Briefly, the immunoblots were probed with anti-mfn1 (ab221661, Abcam), anti-mfn2 (ab205236, Abcam), anti-OPA1 (ab157457, Abcam), anti-Drp1 (ab184247, Abcam), anti-p-Drp1 (Ser637) (ab193216, Abcam), anti-p-Drp1 (ser616) (#3455, Cell Signaling Technology), anti-fis1 (10956-1-AP, Proteintech), anti-C/EBP homologous protein (CHOP) (#2895, Cell Signaling Technology), anti-myosin light chain (MLC) (#3672, Cell Signaling Technology), anti-p-MLC (#3675, Cell Signaling Technology), or anti-β-actin (ab8227, Abcam) overnight at 4° C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL-plus reagent (SignalFire, USA).

Zhuan B., Wang X., Wang M.D., Li Z.C., Yuan Q., Xie J, & Yang Z. (2020). Hypoxia induces pulmonary artery smooth muscle dysfunction through mitochondrial fragmentation-mediated endoplasmic reticulum stress. Aging (Albany NY), 12(23), 23684-23697.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted by RIPA (P0013B, Beyotime, China) with PMSF (AR1192, Boster, China). BCA Protein Assay Kit (P0011, Beyotime, China) were used to quantify proteins. The standard western blotting was performed with primary antibodies against: NRF2 (1:1 000, 16396-1-AP, Proteintech, USA); ACSL4 (1:1 000, 22401-1-AP, Proteintech); FSP1 (1:1 000, 20886-1-AP, Proteintech); GPX4 (1:1 000, ab125066, Abcam, USA); TFRC (1:3 000, ab269513 Abcam); FPN (1:1 000, 26601-1-AP, Proteintech), DMT1 (1:1 000, YN3198, Immunoway, USA); FTH1 (1:1 000, ab183781, Abcam); MFN1 (1:3 000, ab221661, Abcam); MFN2 (1:3 000, ab205236, Abcam); DRP1 (1:3 000, ab184247, Abcam); OPA1 (1:3 000, ab157457, Abcam); Hepcidin (1:1 000, DF6492, Immunoway, USA); GAPDH (1:3 000, 10494-1-AP, Proteintech). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Abcam (1:5 000, Abcam).

Xiang Z., Zhang P., Jia C., Xu R., Cao D., Xu Z., Lu T., Liu J., Wang X., Qiu C., Fu W., Li W., Cheng L., Yang Q., Feng S., Wang L., Zhao Y, & Liu X. (2024). Piezo1 channel exaggerates ferroptosis of nucleus pulposus cells by mediating mechanical stress-induced iron influx. Bone Research, 12, 20.

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Western Blot Analysis of Cellular Proteins

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Western blotting was performed as previously reported. Proteins were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Abcam, Ab156034, United Kingdom). Protein concentration was determined by Pierce™ BCA protein assay kit (Thermo Fisher Scientific, United States of America). Protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat dry milk in Tris-buffered saline, membranes were incubated at 4°C overnight with primary antibodies against mfn2 (ab205236; 1:1,000; Abcam; United States of America), NOX4 (ab 133303; 1:1000; Abcam; United States of America), p53 (ab26; 1:250; Abcam; United States of America), p38 MAPK (ab4822; 1:1000; Abcam; United States of America), and p65 (ab16502; 1:1000; Abcam; United States of America). Subsequently, a secondary goat anti-rabbit IgG (HRP) antibody (Abcam, ab205718, 1:2,000) was applied to incubate with membranes at room temperature for 1 h. The blots were assayed by using the Novex™ ECL chemiluminescent substrate reagent kit (Thermo Fisher Scientific™, WP20005, United States of America). β-actin was used as an internal reference protein. The results were analyzed with ImageJ software.

Li Y., Ou S., Liu Q., Gan L., Zhang L., Wang Y., Qin J., Liu J, & Wu W. (2022). Genistein improves mitochondrial function and inflammatory in rats with diabetic nephropathy via inhibiting MAPK/NF-κB pathway. Acta Cirúrgica Brasileira, 37(6), e370601.

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