Total protein was isolated with a total protein extraction kit (Keygene, Nanjing, China) and quantitated using a BCA assay kit (Keygene). The protein lysates were separated using 8 or 10 % SDS-PAGE gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. After antigen blocking, the transferred PVDF membrane was incubated with primary antibodies for EPHA3 (clone 3A12, 3.5 μg/mL, Abnova, Taiwan), phospho-PIK3R1 (pTyr467, 1:500, Sigma-Aldrich, St. Louis, MO, USA), total PI3K p85α (clone Ab6, 1 μg/mL, Abcam, Cambridge, England), phospho-BMX (Tyr40, 1:1000, Cell Signaling technology), total BMX (Y396, 1:1000, Abcam, Cambridge, England), phospho-Stat3 (Tyr705, D3A7, 1:1000, Cell Signaling technology), total STAT3 (clone 4D6, 10 μg/mL, Abnova), and GAPDH (internal control, 1D4, 1:1000, EarthOx, San Francisco, CA, USA) overnight at 4 °C with gentle shaking. The secondary goat anti-Rabbit-IgG (EarthOx) or goat anti-Mouse-IgG (EarthOx) HRP AffiniPure antibody was added correspondingly at a 1:2000 dilution. After chemiluminescence, the intensity of the protein fragments was quantified using Quantity One software (4.5.0 basic, Bio-Rad Laboratories, Hercules, CA, USA).
Total protein extraction kit
Manufactured by Keygene
Sourced in China
The Total Protein Extraction Kit is a laboratory tool designed for the efficient extraction and isolation of total proteins from various biological samples. This kit provides a standardized protocol and the necessary reagents to obtain a comprehensive protein profile from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Irdye secondary antibody, by LI COR (2 mentions)
Phospho stat3 tyr705 d3a7, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca assay kit, by Keygene (1 mentions)
Goat anti rabbit igg, by EarthOx (1 mentions)
Goat anti mouse igg, by EarthOx (1 mentions)
β actin, by Bio-Techne (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Electrochemiluminescence system, by GE Healthcare (1 mentions)
Ferritin, by Abcam (1 mentions)
Odyssey dual color infrared fluorescence imaging system, by LI COR (1 mentions)
Steap3, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Enhanced chemiluminescence detection kit, by Advansta (1 mentions)
Bicinchoninic acid protein assay kit, by Bio-Rad (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Crosslink magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using total protein extraction kit
1
Comprehensive Protein Analysis Protocol
2
Western Blot Protein Analysis Protocol
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Western Blot assays were performed and analyzed as before (Tang et al., 2020b (link)). Briefly, a whole extract was prepared by a Total Protein Extraction Kit (Keygene Biotech, Nanjing, China). Following normalization of protein concentration by using BCA kits (Thermo Fischer, US), the samples were separated in 10% SDS-PAGE electrophoresis and electrotransferred to a nitrocellulose membrane. Validated antibodies used in this study were: CYP2E1 (1:500 Cat. D122177, Sangon Biotech, China), GPX4 (1:1000, Cat. MAB5457, Bio-techne, USA), β-actin (1:2000, Cat. D110001, Sangon Biotech). The IRDye® secondary antibody (1:10,000, LI-COR, USA) was used and immunoblots were scanned by the Odyssey® dual-color infrared fluorescence imaging system. The grayscale of each band was obtained from Odyssey® software.
3
Protein Expression Analysis in Cells
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Cells were digested with the Total Protein Extraction Kit (KeyGene, China) and centrifuged to collect cell supernatants. A BCA assay was used to determine the protein concentration. Proteins (30 μg) were separated on 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to a PVDF membrane, and then blocked with 5% nonfat dry milk dissolved in TBST (Tris-buffered saline, 0.1% Tween-20). The primary antibodies anti-DMP1, anti-DSPP, anti-OPN, anti-ALP, anti-RUNX2, and GAPDH were used at dilutions of 1:1000 for 2 h at room temperature, and then washed with TBST twice on a TS rocker. Secondary antibodies were applied in the same way. Bands were monitored with an electrochemiluminescence system (GE, USA).
4
Western Blot Analysis of Iron Metabolism Proteins
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Total proteins were extracted by the total protein extraction kit (Keygene Biotech). Western blot assays were performed and analyzed as before.[22 ] Antibodies used in this study were β‐actin (Cat. ab8227; Abcam), SLC46A1 (Cat. ab25134; Abcam), STEAP3 (Cat. ab180770; Abcam), DMT1 (Cat. ab55735; Abcam), TfR1 (Cat. ab84036; Abcam), FPN (Cat. ab85370; Abcam), HAMP (Cat. ab30760; Abcam), and ferritin (Cat. ab75973; Abcam). The IRDye secondary antibody (LI‐COR) was used, and immunoblots were scanned by Odyssey dual‐color infrared fluorescence imaging system. The grayscale of each band was obtained from Odyssey software.
5
Immunoprecipitation and Western Blot Analysis
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Immunoprecipitation assays were performed using the Crosslink Magnetic IP/Co-IP Kit (Thermo Scientific). Cells were solubilized with immunoprecipitation buffer, equal amounts of protein were incubated with specific antibody immobilized onto protein A/G magnetic beads overnight at 4 °C with gentle rotation. Beads were washed extensively with immunoprecipitation lysis buffer, resuspended, and eluted. Protein samples were subjected to SDS-PAGE followed by western blot analysis.
For western blot assay, total protein was extracted from the cultured cells using the total protein extraction kit (KeyGene). The protein content was determined using the bicinchoninic acid protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Proteins (30 μg) were separated by 8–12% SDS–PAGE and transferred onto nitrocellulose filter membranes (Life Science). After blocking in 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20, the membranes were probed with primary antibodies overnight at 4 °C followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at RT. Antigen-antibody complexes were visualized using enhanced chemiluminescence detection kit (Advansta) and image analyzer ImageQuant LAS 500 (GE Healthcare).
For western blot assay, total protein was extracted from the cultured cells using the total protein extraction kit (KeyGene). The protein content was determined using the bicinchoninic acid protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Proteins (30 μg) were separated by 8–12% SDS–PAGE and transferred onto nitrocellulose filter membranes (Life Science). After blocking in 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20, the membranes were probed with primary antibodies overnight at 4 °C followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at RT. Antigen-antibody complexes were visualized using enhanced chemiluminescence detection kit (Advansta) and image analyzer ImageQuant LAS 500 (GE Healthcare).
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