GTPs and EGCG (≥99% purity by high-performance liquid chromatography) were provided by Hangzhou Hetian Biotech Co., Ltd (Hangzhou, Zhejiang Province, China). Total Protein Extraction Kit and BCA Protein Assay Kit were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Antibodies of PKCα, phospho-PKCα (pPKCα), ERK1/2, phospho-ERK1/2 (pERK1/2), PGC-1α and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). The 5% bovine serum albumin (BSA) blocking solution was purchased from Wuhan Boster Bio-engineering Co., Ltd (Wuhan, China). Enhanced Chemiluminescence (ECL) Assay Kit was purchased from Millipore (USA). Cortisol Radioimmunoassay Kit was provided by Beifang Biotech Co., Ltd, (Beijing, China). Dopamine (DA) Enzyme-Linked Immunosorbent Assay (ELISA) Kit and serotonin (5-HT) Enzyme-Linked Immunosorbent Assay (ELISA) Kit were obtained from Shanghai Shiruike Biotech Co., Ltd (Shanghai, China). ATP Assay Kit (ab83355) was purchased from Abcam (Cambridge, MA, USA). Ultrapure RNA Kit was purchased from Beijing Kangwei Century Company (Beijing, China). All-in-one™ First Strand cDNA Synthesis Kit was obtained from Guangzhou GeneCopoeia Co., Ltd, (Guangzhou, China). Other analytical grade chemicals and reagents were purchased from Beijing Chemical Reagent Company (Beijing, China).
Total protein extraction kit
Manufactured by Sangon
Sourced in China
The Total Protein Extraction Kit is a laboratory tool designed to extract and purify total proteins from various biological samples. It provides a standardized procedure for efficient protein extraction and recovery.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (6 mentions)
Bca protein assay kit, by Sangon (5 mentions)
Ab181602, by Abcam (4 mentions)
Protein loading buffer, by Boster Bio (4 mentions)
Anti col3a1 mouse monoclonal antibody, by Santa Cruz Biotechnology (4 mentions)
Goat polyclonal secondary antibody to mouse igg h l hrp, by Abcam (4 mentions)
Dye reagent, by Bio-Rad (4 mentions)
Quick start bovine serum albumin, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Wet transfer system, by Bio-Rad (4 mentions)
Easyblot ecl, by Sangon (3 mentions)
Ab229126, by Abcam (3 mentions)
Ab8448, by Abcam (3 mentions)
Ab236618, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab8245, by Abcam (2 mentions)
β actin, by Proteintech (2 mentions)
Sds page, by Sangon (2 mentions)
Anti atp7a, by Abcam (2 mentions)
30 protocols using total protein extraction kit
1
Molecular Mechanisms of GTP and EGCG in Regulating Cellular Signaling
2
Protein Expression in Dissociated Liver Tissues
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Following liver tissues were routinely dissociated and digested by collagenase type II, the hepatic cells suspensions were collected for extracting total proteins by Total Protein Extraction Kit (Shanghai Sangon Biotech, China) according to the manufacturer’s instructions, then the concentration of proteins was determined using the Bradford reagent (Sigma-Aldrich, USA). The expressions of OX40, Nrf2, NQO1, and inflammatory signaling NOD-like receptor family pyrin domain-containing 3 (NLRP3) were evaluated by the standard steps of Western-blotting (WB) experiment. The primary antibodies were provided as the followings: Anti-CD134/OX40L receptor antibody (ab229021, 1:800, Abcam), anti-Nrf2 antibody (ab92946, 1:1000, Abcam), anti-NQO1 antibody (sc-32,793, 1:800, Santa Cruz Biotechnology), or anti-NLRP3 antibody (ab263899, 1:1000, Abcam). Anti-rabbit IgG-HRP antibody (LS-C86382, LSBio) was used as the secondary antibody. WB results were pictured by enhanced chemiluminescence reagent (Beyotime, China), and then the integrated absorbance (IA) of the bands were analyzed quantitatively using LI-COR Odyssey Imaging System. Anti-GAPDH Rabbit Polyclonal antibody (BM1623, Dingguo Biotech Co. Ltd) were used as an internal control for normalizing the values in equal samples, the relative levels of target proteins were evaluated by the ratio of target protein vs internal control (IA/IA).
3
Total Protein Extraction and Western Blot
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Cell proteins were extracted using the total protein extraction kit (Sangon, Shanghai, China, item no. BSP003), and 5μL phosphatase inhibitor, 1μL protease inhibitor, and 10μL phenylmethanesulfonyl fluoride (PMSF) were mixed in per 1mL precooled lysis buffer. Protein concentration was determined using the BCA assay kit (Sangon, Shanghai, China, item no. SK3021). And Western blot was conducted as previously described.16 (link)
4
Calcineurin Protein Expression in Tianfu Goat Muscles
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Total proteins of five muscle tissues were extracted by using a Total Protein Extraction Kit (Sangon Biotech, Shanghai, China), according to the manufacturer’s protocol, and then the muscle proteins were preserved at −80 °C in a freezer. Western blotting was used to analyze the relative protein expression of PPP3CA in muscle tissues of Tianfu goat at 150 days old. The determination of the protein concentration used BCA protein assay kit (Beyotime, Shanghai, China) with bovine serum albumin (BSA) as a standard. GAPDH protein was selected as an internal control. Polyclonal antibodies to the calcineurin protein (Abcam, Cambridge, UK) were used at 1/500 dilution and a monoclonal antibody to the GAPDH protein (BiYunTian, Shanghai, China) was at a 1/1000 dilution. Equal amounts of protein (12 μg per lane) were resolved on 12% SDS-polyacrylamide gels, and then as detailed previously in [33 (link)], the intensity of the signal was used to analyze the relative amount of PPP3CA protein in Tianfu goat muscle tissues extracts.
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted using a Total Protein Extraction Kit (Sangon, China), and protein concentration was measured with a BCA Protein Assay Kit (Beyotime, China). Protein samples were separated by 10% SDS polyacrylamide gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.
6
Protein Extraction and Western Blot Analysis
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The extraction of total proteins from cultured hepatocellular carcinoma cell lines or mouse liver tissues were done using the Total Protein Extraction kit (#C510003; Sangon Biotech, Shanghai, China) following producer’s protocol. After protein concentration determination by BCA method, about 30 μg protein of each group were boiled at 100 °C for 5 min in protein loading buffer, separated by 10–12% SDS-PAGE, and blotted onto PVDF membranes (Millipore). The PVDF membranes containing protein samples were blocked with lipid-free milk solution (5%) for 1.5 h at room temperature, incubated with diluted primary and secondary antibodies, which was finally developed using the Highly sensitive ECL (enhanced chemiluminescence) luminescence reagent (#C500044; Sangon Biotech, Shanghai, China). The abundances of GAPDH proteins were simultaneously detected as the internal standard. Primary antibodies used for quantitation included anti-MKL1 (#ab49311; 1: 6000; Abcam), anti-P65 (#8242; 1: 5000; CST), anti-SET1 (#61702; 1: 4000; CST), anti-WDR5 (#13105; 1: 5000; CST), anti-ASH2 (#5019; 1: 2000; CST), anti-GAPDH (#ab181602; 1: 5000; Abcam), anti-Bcl-2 (#3498; 1:1000; CST),anti-Bax(#5023; 1:1000;CST), and anti-Cleaved-caspase-3(#9654; 1:1000; CST) .
7
Vladimiria souliei Root Extraction and Evaluation
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The roots of Vladimiria souliei (Franch.) Ling were collected from Luding, Sichuan Province in September of 2015. This plant was identified by Professor Min Chen at Southwest University College of Pharmaceutical Sciences. A voucher specimen (No. CM2015-002) was deposited at the Southwest University College of Pharmaceutical Sciences (Chongqing, China).
LPS (Cat. No. L5293) was obtained from Sigma-Aldrich China, Co., LLC (Shanghai, China).D -GalN was purchased from Aladdin Reagent Database, Inc. (Shanghai, China). Diagnostic kits used for the determination of MDA, CAT, SOD, T-AOC. AST, ALT activities and cytochrome C (Cyto-C) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit IL-6, IL-1β, IκB, p-IκB, IκB, p-IKK, NF-κB, Bax, Bcl-2, Cyto-C, Caspase3, Caspase8, Caspase9, β-actin, mouse TNF-α polyclonal primary antibodies, goat anti-mouse IgG-HRP-conjugated secondary antibody, and goat anti-rabbit IgGHRP-conjugated secondary antibody were obtained from Proteintech Group, Inc. (Wuhan, China), while total protein extraction kit was from Sangon Biotech, Co., Ltd. (Shanghai, China).
LPS (Cat. No. L5293) was obtained from Sigma-Aldrich China, Co., LLC (Shanghai, China).
8
Dyslipidemia Induction in C57BL/6J Mice
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SPF male C57BL/6 J mice aged 7–8 weeks and weighing 20–22 g were obtained from the Chongqing Academy of Traditional Chinese Medicine. Nanjing Jiancheng Bioengineering Institute (Nanjing, China) provided diagnostic kits for the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C). Proteintech Co., Ltd (Wuhan, China) provided rabbit anti-serine/threonine protein kinase 1 (AKT 1), interleukin 6 (IL-6), mitogen-activated protein kinase 1 (MAPK1), cysteinyl aspartate specific proteinase (Caspase 3), p53, TNF-polyclonal primary antibodies from mice, an anti-mouseIgG-HRP-conjugated secondary antibody from goat, and anti-rabbit IgGHRP-conjugated secondary antibody goat. Sangon Biotech Co. Ltd (Shanghai, China) provided the total protein extraction kit. Chongqing Yonghui Supermarket (Chongqing, China) supplied sucrose, lard, and eggs. Dalian Beier Pharmaceutical Co., Ltd (Dalian, China) provided cholesterol, sodium cholate, and propylthiouracil.
9
Quantitative Western Blot Analysis
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Total proteins extracted using a total protein extraction kit (Sangon Biotech) were quantified using a BCA protein assay kit (Sangon Biotech). Equal amount of protein was separated and transferred into PVDF membrane (Bio-Rad). The membranes containing the isolated proteins were blocked in 5% skim milk, exposed to the primary antibodies targeting LMO4 (ab229226; Abcam, Cambridge, MA, USA) and GAPDH (ab8245; Abcam) at 4°C overnight, and next reacted with the secondary antibody (ab205718; Abcam) for 2 h at room temperature. The indicated protein signals were presented using the enhanced chemiluminescence kit (Sangon Biotech) and quantified using the Image J software.
10
Placental Protein Extraction and Western Blot Analysis
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Placental tissue was weighed and then processed with a total protein extraction kit
(Sangon Biotech, China) according to the manufacturer's instructions. Protein samples
were resolved by polyacrylamide gel electrophoresis on 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gels and electrotransferred to polyvinylidene fluoride (PVDF)
membranes. After blocking with Tris-buffered saline with Tween (TBS-T) with 5%
non-fat milk for 2 h at room temperature, membranes were incubated with the vaspin
polyclonal antibody (1:1000, GeneTex, USA) overnight at 4°C. The immunoreactive bands
were detected by a chemiluminescence detection kit (Tiangen, China) after incubation
with horseradish peroxidase-labeled rabbit IgG antibody (1:10,000, Proteintech, USA).
The membrane was re-probed with β-actin (1:5000, Proteintech) as a protein loading
control. The results were analyzed with a White/Ultraviolet Transilluminator (UV-II,
Gene Company Limited, China).
(Sangon Biotech, China) according to the manufacturer's instructions. Protein samples
were resolved by polyacrylamide gel electrophoresis on 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gels and electrotransferred to polyvinylidene fluoride (PVDF)
membranes. After blocking with Tris-buffered saline with Tween (TBS-T) with 5%
non-fat milk for 2 h at room temperature, membranes were incubated with the vaspin
polyclonal antibody (1:1000, GeneTex, USA) overnight at 4°C. The immunoreactive bands
were detected by a chemiluminescence detection kit (Tiangen, China) after incubation
with horseradish peroxidase-labeled rabbit IgG antibody (1:10,000, Proteintech, USA).
The membrane was re-probed with β-actin (1:5000, Proteintech) as a protein loading
control. The results were analyzed with a White/Ultraviolet Transilluminator (UV-II,
Gene Company Limited, China).
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