In this study, proteins were extracted using a protein extraction kit (Sangon Biotech), and the bicinchoninic acid (BCA) assay (Sangon Biotech) was used to determine the total protein content. After denaturation for 5 min, total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA) via a constant current flow at 200 mA. Subsequently, the PVDF membranes were incubated with Bcl-2 (1:10,000), Bax (1:2000), Caspase-3 (1:5000), M-CSF (1:1000), RANKL (1:3000), OPG (1:3000), p-p65 (1:1000), p65 (1:1000), p-p38 (1:1000), p38 (1:5000), p-JNK (1:1000), JNK (1:1000), p-AKT (1:2000), AKT (1:2000), p-ERK (1:1000), ERK (1:1000), RANK (1:2000), NF-κB (1:1000), NFATc1 (1:10,000), and c-Fos (1:1000) antibodies (Abcam, Cambridge, MA) for 12 h at 4°C. TBS buffer was used to wash the PVDF membranes, and secondary antibodies (Abcam) were added and incubated at room temperature for 1 h. After the membranes were washed three times, chemiluminescent reagents were added, and the grayscale values of the bands were analyzed using ImageJ software. Each experiment was independently repeated 3 times. GAPDH was used to quantify the expression of various proteins.
Protein extraction kit
Manufactured by Sangon
Sourced in China
The Protein Extraction Kit is a laboratory tool designed to efficiently isolate and purify proteins from various biological samples. It provides a standardized method for extracting proteins while preserving their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Protein quantitation kit, by Bio-Rad (2 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay kit, by Sangon (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
P akt, by Abcam (1 mentions)
Bca assay, by Sangon (1 mentions)
P jnk, by Abcam (1 mentions)
P erk, by Abcam (1 mentions)
Nfatc1, by Abcam (1 mentions)
C fos, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Nf κb, by Abcam (1 mentions)
Rankl, by Abcam (1 mentions)
M csf, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Protein g plus agarose beads, by Thermo Fisher Scientific (1 mentions)
15 protocols using protein extraction kit
1
Protein Expression Analysis Protocol
2
Protein Complex Identification and Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates and cytoplasmic protein were prepared following the instructions of a protein extraction kit from Sangon Biotech (C006255 and C510001). For western blot analysis, 30 µg of whole protein lysates was used to detect the indicated protein, 4-12% NuPAGE Bis-Tris gel (NP0322BOX, Invitrogen) was used for electrophoresis. STRING database (https://string-db.org ) was used for predicting the protein combination before experiments. For Co-IP assay, 1% Triton (strong lysis buffer which can depolymerize the mTOR complex) or CHAPS (mild buffer which can maintain the integrated mTOR complex) lysis buffer was used. After pre-incubation with protein G PLUS-Agarose beads (20423, Thermo Fisher Scientific), an equal amount of protein (500 µg) was incubated with the indicated antibodies (1 µg, RICTOR or TELO2). 1% Triton or CHAPS buffer was used to wash beads at 4°C, 200 × g three times. Then 1% of the input was loaded to detect the protein level. PVDF (LC2002; Thermo Fisher) was used as transmembrane. ImageJ software was used to scan the grey scores of images. Cycloheximide (CHX; Calbiochem) was used for protein chasing experiment.
3
Western Blotting Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting assays were performed as described [38 (link),39 (link)]. The liver and hepatocyte protein were extracted using a protein extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The protein concentration was measured using protein assay reagent (Sangon Biotech). The target proteins were separated in polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a 3% BSA in 0.1% Tris-buffered saline and Tween (TBST) buffer for 4 h. The membranes were hybridized with antibodies specific for PGC-1α (1:1000), Akt (1:1000), GSK-3β (1:1000), P-Akt (1:1000), P-GSK-3β (1:1000), NDUFA9 (1:1000), SDHA (1:1000), UQCRC2 (1:1000), COXIV (1:1000), ATPB (1:1000) (Abcam, USA) and β-Actin (1:5000) (ZSGB-BIO, Beijing, China) overnight at 4 °C. The PVDF membranes were incubated with appropriate peroxidase-conjugated secondary antibodies for 45 min. Immunoreactive bands were detected with enhanced chemiluminescence solution (Beyotime Biotech). The blots were normalized to β-Actin and quantified via densitometry using Image-Pro Plus 6.0 (Media Cybermetics, Rockville, MD, USA). Experiments were carried out in triplicate.
4
Protein Expression Analysis in Skin Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were obtained from the skin tissues and melanocytes according to the Protein Extraction Kit (Sangon Biotech., China). Protein content was quantified using the Bio-Rad protein assay. Then, samples were diluted, heated for denaturation, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA) and blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk for 1 h. Then, 1:1,000 dilutions of rabbit polyclonal primary antibody anti-mc1r-C (HuaAn Biotechnology Co., Ltd., China) or anti-β-actin (loading control) were incubated at 4°C overnight. The following day, the membrane was rinsed in TBST three times and incubated with a fluorescent secondary anti-rabbit antibody (1:5,000) for 1 h at 37°C. After washing three times with Tris-buffered saline at 5 min per wash, the proteins were detected using ECL chemiluminescence (Beyotime, China) by image software (Bio-Rad Laboratories, PA, USA).
5
Protein Expression in Ovarian Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At least three ovaries were homogenized in the lysis buffer from the protein extraction kit (Sangon Biotech, Shanghai, China). A protein quantitation kit (Bio-Rad) was used to measure the concentrations of the protein solutions. Briefly, the protein samples were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were blocked with 5% defatted milk in Tris-buffered saline containing Tween20 (TBST) for 1 h at room temperature. Next the proteins were incubated with primary antibodies- GRP78 (1:1000, Cell Signaling Technology, Inc.), ATF4 (1:1000, Cell Signaling Technology, Inc.), CHOP (1:1000, Cell Signaling Technology, Inc.), phosphor-JNK (1:1000, Cell Signaling Technology, Inc.), Bax (1:1000, Proteintech, Inc.), Bcl-2 (1:1000, Proteintech, Inc.), and cleaved caspase 3 (1:1000, Cell Signaling Technology, Inc.), overnight at 4 °C respectively. Anti-GAPDH monoclonal antibody (1:2000, Proteintech, Inc.) was used as a loading control. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, the proteins were detected by enhanced chemiluminescence detection system (Bio-Rad).
6
Quantifying TGF-β1 in Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein in the right lungs was extracted using a Protein Extraction kit (Shanghai Sangon Biotechnology, Shanghai, China). Protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Shanghai Sangon Biotechnology). Concentrations of transforming growth factor (TGF)-β1 in lung homogenates were quantified with a Mouse TGF-β1 protein extraction and enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. Optical density values were detected using a Microplate Reader (iMark; Bio-Rad, Hercules, CA, USA) at 450 nm.
7
Quantitative Analysis of p62 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extracted from the ovarian tissue in the estrus phase were obtained using a protein extraction kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instructions without any modifications. Following denaturation with reducing agents and SDS, these proteins were separated on a polyacrylamide gel, subsequently transferred to PVDF membranes (Bio-Rad Laboratories), and blocked with 5% skim milk (Servicebio) for 1 h at room temperature. The membrane was initially incubated with p62 primary antibody (1:1000, Cell Signaling Technology, #5114) overnight at four degrees Celsius, followed by incubation with a secondary antibody conjugated to a detection enzyme for 1–2 h. The protein was then detected by exposing the membrane to a chemiluminescence detection system (Bio-Rad, CA, USA). The bands’ intensity was quantified using Image J software, and the results were compared between samples.
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from the cell was collected using a commercial Protein Extraction Kit (Sangon, Shanghai, China), following the manufacturer’s protocol. The concentration of the protein was measured using the Bradford protein assay kit (Beyotime, Jiangsu, China), and only protein meeting quality criteria were used for the further trial. Briefly, the protein was resolved on 10% SDS-PAGE and then transferred to a PVDF membrane, followed by sealing of the sealing fluid. The membranes were incubated with the correspondingly primary antibodies for 24 h at 4 °C and subsequently incubated with the secondary antibodies for 2 h. The membranes were subjected to chemiluminescence reagents to detect immunoreactivities. A GelDoc system equipped (Bio-Rad, Hercules, CA, USA) was used to capture images. β-actin protein was used as an internal control.
9
Western Blot Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissue and hepatocyte total proteins were extracted using a protein extraction kit (C510003; Sangon Biotech Co., Ltd., Shanghai, China). Nucleus and cytoplasm separation were conducted using a Nuclear-Cytosol Extraction Kit (P1200, Applygen Technologies). The protein concentration was estimated by the BCA method (P1511; Applygen Technologies). The samples were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane. The membranes were blocked in 3% BSA/Tris-buffered saline/Tween buffer for 4 h. The blocked membrane was incubated overnight at 4 °C with the primary antibody. The membranes were then incubated with HRP-conjugated anti-rabbit or anti-mouse IgG at room temperature for 45 min. Immunoreactive bands were visualized by enhanced chemiluminescence solution (WBKLS0500, Millipore, Bedford, MA, USA). All bands were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). The Western blots shown are representative of 3 independent experiments with consistent results.
10
Quantifying Brain Cytokine Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We sacrificed the mice on day 3 post-surgery and prepared the brain homogenates for ELISA as previously described [31 (link)]. We homogenized the right hemisphere using a protein extraction kit (Sangon Biotech, Shanghai, China) and measured protein concentration with a bicinchoninic acid protein assay kit (Sangon Biotech). We adjusted the total protein concentration to 1 mg/ml protein extract and quantified the concentrations of TNF-α and IL1β in brain homogenates according to the manufacturer’s protocol (Boster, Wuhan, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!