Radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) was used for the extraction of proteins, and the bicinchoninic acid (BCA) method (Beyotime Institute of Biotechnology) was used for the measurement of concentrations. Proteins (40 µg/lane) were loaded on 8% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked for nonspecific binding for 2 hours by incubation with 5% fat-free dry milk in 100 mM of Tris-buffered saline plus 0.1% Tween-18 (TBST). Primary antibodies included rabbit anti-NEDD4L antibody (ab46521; 1:1,000; Abcam), rabbit anti-ENaC-α antibody (ab214192; 1:1,000; Abcam), and rabbit anti-GAPDH antibody (KGAA002; 1:1,000; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The membrane was incubated with primary antibodies overnight at 4 ℃ and then washed with TBST 3 times. The membrane was then incubated with HRP-conjugated goat-anti-rabbit secondary antibody (bs-0295G-HRP; 1:2,000; Bioss, Woburn, MA, USA) for 1.5 hours at room temperature. The enhanced chemiluminescence detection system (ChemiDoc XRS + Imaging System; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to visualize the bands of antibody-detected proteins.
Kgaa002
Manufactured by Keygen Biotech
Sourced in China
The KGAA002 is a high-performance gel electrophoresis system designed for the separation and analysis of DNA, RNA, and protein molecules. It features a stable and uniform electric field, ensuring accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab46521, by Abcam (1 mentions)
Ab214192, by Abcam (1 mentions)
Bs 0295g hrp, by Bioss Antibodies (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Bca method, by Beyotime (1 mentions)
Gel pro analyzer version 4, by Media Cybernetics (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab31163, by Abcam (1 mentions)
Kga902, by Keygen Biotech (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
Ab62352, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
4 protocols using kgaa002
1
Western Blot Analysis of NEDD4L, ENaC-α, and GAPDH
2
TM6SF2 Regulates Inflammatory Cytokines
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Western blotting was used to test the effects of TM6SF2 or TM6SF2 E167K on the protein expression of TNF-α, IL-2, IL-6, and IL-8. HEPA 1-6 cells were collected after infection with the lentiviral solution for 48 h, providing samples for overexpressed TM6SF2, TM6SF2 E167K expression, or negative control. RIPA buffer (Sigma-Aldrich, USA) was used to extract total protein from the HEPA 1-6 cells. Protein concentrations were determined by the Bradford method. Antibodies against TNF-α (KG22761; Jiangsu Keygen Biotech Co., Ltd., China), IL-2 (KG22463; Jiangsu Keygen Biotech Co., Ltd.), IL-6 (KG22468; Jiangsu Keygen Biotech Co., Ltd.), IL-8 (KG22469; Jiangsu Keygen Biotech Co., Ltd.) and GAPDH (KGAA002; Jiangsu Keygen Biotech Co., Ltd.) were used. The expression levels of TNF-α, IL-2, IL-6 and IL-8 proteins were normalized relative to the corresponding GAPDH (endogenous reference) level in each lane. The western blots were analyzed using Gel-Pro Analyzer Version 4.5 software (Media Cybernetics, USA).
3
Western Blot Analysis of Antioxidant Proteins
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Lung tissues were homogenized and incubated in lysis buffer containing a protease inhibitor cocktail. The protein concentrations were measured using a BCA kit (KGA902, KeyGEN BioTECH, Nanjing, China), and proteins were then denatured at 100 °C for 5 min. Proteins were loaded onto a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Blots were blocked at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. The details of primary antibodies used in this paper were shown as follows: rabbit anti-GPX4 (14432–1-ap, Proteintech, 1:500), rabbit anti-Nrf2 (ab31163, abcam, 1:500), mouse anti-HO1 (sc-136,960, santa, 1:200), and mouse anti-NQO1 (sc-32,793, santa, 1:200), rabbit anti-GAPDH (KGAA002, KeyGEN BioTECH, China, 1:10000). After washing, blots were incubated with fluorescence-labeled secondary antibodies.
4
Western Blot Analysis of Oxidative Stress Markers
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Cells were lysed in RIPA buffer containing 1 nM phenylmethylsulfonyl fluoride (PMSF) and phosphatase and protease inhibitors on ice. Then, the lysates were centrifuged at 12,000 g for 20 min at 4°C, and the protein concentration of the supernatant was determined using the BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein were separated via 8% to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-coupled secondary antibodies incubation. The details of primary antibodies used in this research were shown as follows: P62 (1 : 10000, ab155282, Abcam, UK), Keap1 (1 : 500, 10503-2-AP, Sanying BioTECH, China), Nrf2 (1 : 1000, ab62352, Abcam, UK), HO1 (1 : 500, 10701-1-AP, Sanying BioTECH, China), NQO1 (1 : 500, 11451-1-AP, Sanying BioTECH, China), FTH1 (1 : 1000, ab170888, Abcam, UK), and rabbit anti-GAPDH (1 : 10000, KGAA002, KeyGEN BioTECH, China). The immunocomplexes were subsequently detected on photographic film using a chemiluminescence reagent (Millipore, Bedford, MA, USA).
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