Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), and lyophilized trypsin-EDTA were obtained from GIBCO BRL (Grand Island, NY, USA). 3-[4,5-Dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), and Tris/HCl were purchased from Sigma (St. Louis, MO, USA). The whole protein extraction kits were purchased from Keygen (Keygen Biotech. Co. Ltd., Nanjing, China). Trizol reagent, PrimeScript RT Master Mix, and SYBR Green PCR Master Mix reagent kits were obtained from TaKaRa (TaKaRa Biotechnology, Dalian, China). The primers were synthesized by Invitrogen Life Tech (Carlsbad, CA, USA). Rabbit monoclonal anti-AKT (phospho S473), rabbit polyclonal anti-Plzf ab38739, and anti-Oct-4 ab18976 were from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-β-actin was from Chemicon (Temecula, CA). Enhanced chemiluminescence was obtained from Amersham Biosciences (Uppsala, Sweden).
Whole protein extraction kit
Manufactured by Keygen Biotech
Sourced in China
The Whole Protein Extraction Kit is a laboratory tool designed to efficiently extract and isolate complete protein molecules from biological samples. The kit provides a standardized protocol and the necessary reagents to facilitate the extraction process, enabling researchers to obtain high-quality, intact protein samples for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Primescript rt master mix, by Takara Bio (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Enhanced chemiluminescence, by Cytiva (3 mentions)
Sybr green pcr master mix reagent kit, by Takara Bio (3 mentions)
Anti akt phospho s473, by Abcam (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Tris hcl, by Merck Group (3 mentions)
Lyophilized trypsin edta, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Abcam (3 mentions)
Bca protein assay kit, by Keygen Biotech (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
3 4 5 dimethylthiazolyl 2 2 5 diphenyltetrazolium bromide mtt dimethyl sulfoxide dmso sodium dodecyl sulfate, by Merck Group (2 mentions)
Rabbit polyclonal anti plzf ab38739, by Abcam (2 mentions)
Anti oct4 ab18976, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
E cadherin, by Abcam (2 mentions)
46 protocols using whole protein extraction kit
1
Cytotoxicity and Gene Expression Analysis
2
Glial Cell-Derived Neurotrophic Factor Assay
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Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and lyophilized trypsin-ethylenediaminetetraacetic acid were purchased from GIBCO BRL (Grand Island, NY, USA). 3-[4,5-Dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT), diethyl pyrocarbonate, dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), ammonium peroxodisulphate, and Tris-hydrochloride were obtained from Sigma (St. Louis, MO, USA). The recombinant murine GDNF was purchased from Pepro Tech (Rocky Hill, NJ, USA). TRIzol reagent, PrimeScript RT Master Mix, and SYBR Green PCR Master Mix reagent kits were obtained from TaKaRa (TaKaRa Biotechnology, Dalian, China). The primers were synthesized by Invitrogen Life Technologies (Carlsbad, CA, USA). The whole protein extraction kits were purchased from KeyGen (KeyGen Biotech. Co. Ltd., Nanjing, China). The goat polyclonal anti-POU3F1 and rabbit polyclonal anti-GFRα1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit monoclonal anti-AKT (phospho S473) was purchased from Abcam (Cambridge, MA, USA). The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from BioWorld (St. Louis Park, MN, USA). Enhanced Chemiluminescence was obtained from Amersham Biosciences (Uppsala, Sweden).
3
Cytotoxicity and Gene Expression Analysis
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Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), and lyophilized trypsin-EDTA were obtained from GIBCO BRL (Grand Island, NY, USA). 3-[4,5-Dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), and Tris/HCl were purchased from Sigma (St. Louis, MO, USA). The whole protein extraction kits were purchased from Keygen (Keygen Biotech. Co. Ltd., Nanjing, China). Trizol reagent, PrimeScript RT Master Mix, and SYBR Green PCR Master Mix reagent kits were obtained from TaKaRa (TaKaRa Biotechnology, Dalian, China). The primers were synthesized by Invitrogen Life Tech (Carlsbad, CA, USA). Rabbit monoclonal anti-AKT (phospho S473), rabbit polyclonal anti-Plzf ab38739, and anti-Oct-4 ab18976 were from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-β-actin was from Chemicon (Temecula, CA). Enhanced chemiluminescence was obtained from Amersham Biosciences (Uppsala, Sweden).
4
Inflammatory Signaling Pathway Modulation
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The RAW264.7 cells were seeded in a 6‐well plate (106 cells per well). After the treatment with the tFNAs and the addition of LPS and ATP, protein samples were obtained from the different groups using whole‐protein extraction kits (KeyGen Biotech Co., Ltd.). The primary antibodies (i.e., anti‐β‐actin, anti‐NF‐κB‐p65, anti‐NF‐κB‐p‐p65, anti‐Nrf2, anti‐NLRP3, anti‐GSDMD, anti‐GSDMD N‐terminal, anti‐caspase‐1, anti‐cleaved caspase‐1, anti‐IL‐1β and anti‐IL‐18) were purchased from Abcam. After incubation for 24 h with the primary antibodies (dilution ratio according to the manufacturer's protocol), the protein samples were incubated with secondary antibodies (1:2000) over a period of 1 h, wherein they were washed 3 times (5 min per wash) with Tris Buffered Saline with Tween (TBST) solution (1×) between each step. Enhanced chemiluminescence (ECL) detection (Bio‐Rad) was performed to detect the protein bands, and the semi‐quantitative analysis of the results was carried out using the ImageJ software.
5
Exosomal Protein Profile Analysis
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Proteins in exosomes or cells were lysed and extracted using a whole-protein extraction kit (KeyGEN, Nanjing, China). Then, the proteins were separated by SDS–PAGE and transferred to NC membranes, blocked in 5% skim milk at room temperature for 1.5 h, and incubated with the following primary antibodies: anti-CD9 (1:300; Abcam, Cambridge, MA, USA), anti-CD63 (1:300; Abcam, Cambridge, MA, USA), anti-TSG101 (1:300; Abcam, Cambridge, MA, USA), anti-calnexin (1:300, Abcam, Cambridge, MA, USA), anti-TNFα (1:1,000; Abcam, Cambridge, MA, USA), anti-IL-6 (1:1000; Abcam, Cambridge, MA, USA), anti-ALP (1:1,000, Boaosen, China), anti-RUNX2 (1:1,000; Abcam, Cambridge, MA, USA), anti-COL-1 (1:1,000; Abcam, Cambridge, MA, USA), and anti-GAPDH (1:5,000; Abcam, Cambridge, MA, USA). The blot was then stained with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (KGAA35; KeyGEN, Nanjing, China). Finally, the protein expression level was detected using a chemiluminescence detection system. Each experiment was repeated 3 times.
6
Western Blotting Protocol for Protein Analysis
7
Steroidogenesis Pathway Regulation Protocol
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RPMI 1640 medium, fetal bovine serum (FBS), and lyophilized trypsin-EDTA were obtained from GIBCO BRL (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), human chorionic gonadotropin (hCG), diethylpyrocarbonate (DEPC), sodium dodecyl sulfate (SDS), and Tris/HCl were purchased from Sigma (St. Louis, MO, USA). Whole protein extraction kit was purchased from Keygen Biotech Co. Ltd. (Nanjing, China). PrimeScript RT Master Mix and SYBR Green PCR Master Mix reagent kits were obtained from TaKaRa (TaKaRa Biotechnology, Dalian, China). The primers used in this study were synthesized by Invitrogen Life Tech (Carlsbad, CA, USA). The BCA Protein Assay kit was purchased from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). PageRuler Prestained Protein Ladder was obtained from Thermo Fisher Scientific (Thermo Fisher Scientific, Shanghai, China). Rabbit polyclonal anti-Nur77, rabbit polyclonal anti-HSD3B, mouse polyclonal anti-StAR, and mouse polyclonal anti-GAPDH antibodies were purchased from Abcam (Cambridge Science Park, Cambridge, UK). HRP-conjugated secondary antibodies were from Jackson ImmunoResearch (Baltimore, PA, USA). Enhanced chemiluminescence kit was obtained from Amersham Biosciences (Uppsala, Sweden).
8
Western Blot Analysis of Apoptosis and EMT Markers
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Total protein was extracted using the Whole Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentration was quantified using an Enhanced BCA Protein Assay kit (Beyotime, Shanghai, China). Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with primary antibodies against Bcl-2 (1:1,000; Abcam, Cambridge, MA, USA), Bax (1:1,000; Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:2,000; Abcam), N-cadherin (1:2,000; Abcam), Vimentin (1:2,000; Abcam), Slug (1:1,000; Cell Signaling Technology), Twist (1:1,000; Abcam) or GAPDH (1:1,000; Cell Signaling Technology). The protein bands were then probed with HRP-conjugated secondary antibodies for 1 hr at room temperature, visualized using an enhanced chemiluminescence reagent (Millipore) and quantified using Image J 3.0 software (http://imagej.nih.gov/ij/
9
Cultivation and Characterization of Renal Epithelial Cells
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The human renal proximal tubular epithelial (HK-2) and human-derived renal epithelial (HEK-293) cells from the Procell Life Science and Technology (catalog nos. CL-0109 and CL-0005) were used. HK-2 and HEK-293 cells were maintained in respective DMEM/low glucose and DMEM/high glucose supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (50 units/mL), and streptomycin (50 μg/mL) at 37°C in 5% CO2. Primary antibodies against Erk1/2, p38 MAPK, Akt (panspecific antibody and active form), and GAPDH were purchased from Cell Signaling Technology Inc. The DyeLight 680-labeled secondary antibody to rabbit IgG (H + L) was a product of LKP, Inc. BCA Protein Assay Kits were purchased from Thermo Inc. The whole protein extraction kit was from KeyGEN BioTECH.
10
Protein Expression Analysis in NPCs
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The total protein content of NPCs was obtained using a whole protein extraction kit (Nanjing KeyGen Biotech, Nanjing, China), and equal amounts of protein samples were mixed with a sample buffer (Beyotime) and boiled for ten minutes. Protein samples were separated with 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies for A20 (1:2,000; Cell Signaling), p53 (1:1,000; Cell Signaling), p16 (1:1,000; Abcam, Cambridge, UK), aggrecan (1:1,000; Abcam), collagen II (1:5,000; Abcam), NF-κB (1:1,000; Cell Signaling), phosphorylated (p-)NF-κB (1:2,000; Abcam), and β-actin (1:2,000; Abcam) overnight at 4°C. Afterwards, the membranes were incubated with HP-conjugated secondary antibody for two hours at room temperature. Protein bands were visualized using a SuperSignal West Pico Trial Kit (Thermo Fisher). Bands were detected and assessed through Grayscale analysis.
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