Cells were prepared as the protocol described previously [28 (link)]. Fresh liver tissues isolated from mice with or without HFD, GRb1, and GW9662 were digested by collagenase for 30 min at 37°C. Then, cells were filtered and centrifuged. Cells were used for protein extraction from the nucleus and cytoplasm with a kit (cat no. KGP1100; KeyGEN BioTECH Corp., Ltd., Jiangsu, China) according to the manufacturer's protocol.
Kgp1100
Manufactured by Keygen Biotech
Sourced in China
The KGP1100 is a high-precision laboratory instrument designed for performing DNA and RNA extraction and purification. The core function of the KGP1100 is to isolate and concentrate nucleic acids from various biological samples, including cells, tissues, and bodily fluids. The device utilizes advanced extraction techniques to ensure efficient and reliable sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab115730, by Abcam (1 mentions)
Goat anti mouse igg, by R&D Systems (1 mentions)
P0033, by Beyotime (1 mentions)
Haf007, by R&D Systems (1 mentions)
Histone h3, by Santa Cruz Biotechnology (1 mentions)
Glut1, by Abcam (1 mentions)
Sw3015, by Solarbio (1 mentions)
Pc0020, by Solarbio (1 mentions)
Ab89443, by Abcam (1 mentions)
Ab11079, by Abcam (1 mentions)
Ma1 13029, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
P0013, by Beyotime (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
4 protocols using kgp1100
1
Isolation and Fractionation of Hepatocytes
2
Subcellular Protein Isolation and Western Blot Analysis
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Nuclear, cytoplasmic (CYT) and plasma membrane (PM) protein of PFC tissues or astrocytes were isolated by commercial kits (KGP1100, KeyGEN BioTECH; P0033, Beyotime Biotechnology). Phenylmethanesulfonyl fluoride (PMSF, a protease inhibitor) was used to inhibit protein degradation. The protein concentration of sample was detected by bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Lysates were loaded into 10% sodium dodecyl sulfate-polyacrylamid gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% non-fat milk for 1 h. Then, the following first antibodies were used: GLUT1 (1:5000, ab115730, Abcam), TXNIP (1:1000, #14715, CST), Na, K ATPase (1:1000, #3010, CST), GR (1:1000, #3600, CST), Histone H3 (1:500, sc-517576, Santa Cruz) and β-actin (1:2000, #4970, CST). After incubating with first antibody overnight, membranes were incubated goat anti-mouse IgG (1:5000, HAF007, R&D systems) or goat anti-rabbit IgG (1:3000, #7074, CST) for 1.5 h. Protein bands were visualized by ECL (#180–5001, Tanon) and imaged by chemiluminescence imaging instrument (#5200, Tanon). The gray value of images was measured by ImageJ.
3
Western Blot Analysis of Tissue Proteins
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Cell and lung tissue proteins were obtained with a protein extraction kit (KGP1100; Keygen Biotech,) and measured with a BCA test kit (PC0020; Solarbio). The 30ug protein was gently loaded to 10% SDS‐PAGE, transferred to the PVDF membrane, and blocked with 5% BSA (SW3015; Solarbio) for an hour. The membrane was soaked with corresponding primary antibodies against OPN, p‐Akt, Akt, p‐ERK, ERK, tumour necrosis factor‐alpha (TNFα), and GAPDH (Table S1 ) at 4°C overnight. Horseradish peroxidase‐conjugated corresponding secondary antibodies were immersed for an hour. Bands were shown using ECL and quantified using ImageJ software.
4
Quantification of Nrf2 and Glo1 Proteins
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To obtain total protein samples, frozen samples of kidney cortex were homogenized in ice‐cold radioimmunoprecipitation assay lysis buffer (P0013; Beyotime, Beijing, China) with 1% protease inhibitors cocktail and phenylmethane sulfonyl fluoride. The protein concentration of the supernatants was determined by the bicinchoninic acid assay kit and adjusted to the same level in all samples. The separation of nuclear and cytosol protein was carried out according to the manufacturer's instructions (KGP1100; KeyGEN BioTECH, Beijing China).
The method of western blot has been previously reported elsewhere24 . The membrane was incubated overnight with a primary mouse anti‐Nrf2 antibody (1:1,000 ab89443; Abcam, Cambridge, UK) or mouse anti‐Glo1 antibody (1:3,000, MA1‐13029; Invitrogen, Waltham, MA, USA) at 4°C. After washing, the membrane was incubated with a secondary antibody (1:5,000 HRP‐coupled anti‐mouse antibody, ZB‐2305; ZSGB‐BIO, Beijing, China) for 1 h at room temperature. The membrane was probed for β‐actin (1:3,000, TA‐09; ZSGB‐BIO) as a loading control for total protein or cytoplasmic protein. Histone was used as a loading control for nuclear protein (1:3,000, ab11079; Abcam). The bands were visualized using the ECL Western Blotting Substrate (Thermo Fisher, Hudson, NY, USA), and quantified with Image‐Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
The method of western blot has been previously reported elsewhere
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