Cells were washed with PBS and harvested using the RIPA buffer (Beyotime, Nanjing, China). Protein concentration was determined by BCA assay (Beyotime, Nanjing). Total protein (20 µg) was separated using 12% SDS-PAGE, transferred to a nitrocellulose membrane and blocked with 5% milk at room temperature for 1 h. After denaturation, the total protein was separated using SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Nanjing). After blocking the proteins with 5% skimmed milk (Beyotime, Nanjing) for 1 h, the PVDF membranes were incubated with rabbit anti-mouse monoclonal antibodies against TIMELESS (1:1000 dilution; cat. no. 109512; Abcam), ESPL1 (1:500 dilution; cat. no. PAB0608; Abnova, Taipei, China) and GAPDH (1:1000 dilution; cat. no. 8245; Abcam, Cambridge, UK) overnight at 4 °C in a shaking incubator. The membranes were then washed with Tris-buffered saline containing Tween-20 and incubated with the respective anti-rabbit IgG secondary antibodies (1:2000 dilution; cat. no. 150077; Abcam) for 1.5 h at room temperature. The immunoblots were visualized using the Odyssey Infrared Imaging System (Li-COR Biosciences, Inc., Lincoln, NE, USA).
Skimmed milk
Manufactured by Beyotime
Sourced in China, United States
5% skimmed milk is a milk product that has had a portion of its fat content removed, resulting in a lower fat concentration of approximately 5%. This product is designed for use in laboratory settings and may serve as a reference or control sample in various experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ecl kit, by Beyotime (3 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Hrp conjugated secondary anti igg antibody, by Beyotime (2 mentions)
Hrp substrate luminol reagent, by Merck Group (2 mentions)
Gel doc ez imager, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca kit, by Beyotime (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Bca assay, by Beyotime (1 mentions)
Gapdh, by Abnova (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Detergent compatible bradford protein quantification kit, by Vazyme (1 mentions)
16 protocols using skimmed milk
1
Western Blot Analysis of Cell Proteins
2
Western Blot Analysis of PDIA6 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T24T and EJ cells were collected at 48 h post-transfection and resuspended in RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentration of protein was determined using a bicinchoninic acid Protein Assay kit (Beyotime Institute of Biotechnology). In total, 50 µg of each protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Following blocking with 5% skimmed milk (Beyotime Institute of Biotechnology) for 2 h at room temperature, the membranes were incubated with primary antibodies against PDIA6 (1:2,000; cat. no. ab227545; Abcam) or GAPDH (1:5,000; cat. no. ab9485; Abcam) at 4˚C overnight. After being washed three times in TBS-T (0.1% Tween-20), the membrane was incubated with the HRP-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (1:10,000; cat. no. ab97051; Abcam) at room temperature for 1 h before developing with an ECL kit (Beyotime Institute of Biotechnology). Data analysis was performed using ImageJ Software version 1.8.0 (National Institutes of Health) to evaluate the expression levels of proteins.
3
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from samples were isolated using RIPA buffer (Vazyme). The protein concentration was checked by Detergent Compatible Bradford Protein Quantification Kit (Vazyme). Proteins were segregated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China) which were blocked with 5 % skimmed milk (Beyotime). Afterward, the membranes were washed by Phosphate-buffered saline (PBS) and then incubated with the primary antibodies: anti-TRIB2 (1:1500, ab84683,Abcam, Cambridge, United Kingdom), anti-β-catenin (1:3000, ab32572, Abcam), anti-c-Myc (1:2500, ab32072, Abcam), anti-E-cadherin (1:3000, ab40772, Abcam) or anti-β-actin (1:3000, ab8227, Abcam)overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being treated with ECL kit (Beyotime).
4
METTL3 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer (Beyotime, Shanghai, China) was used to lyse the cells on ice which were then centrifuged 12,000× g at 4 °C for 15 min. The BCA protein determination kit (Beyotime, Shanghai, China) was used to determine the protein content. The proteins were isolated by 10% SDS-PAGE and transferred to PVDF membranes (Merck-Millipore, Darmstadt, Germany). Membranes were blocked with 5% skimmed milk (Beyotime, Shanghai, China) for 1 h and probed overnight with anti-METTL3 antibody (1:1000, Cat.#E3F2A, Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibody (1:1000, Cat.#AC001, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight, and followed by incubation with HRP-conjugated secondary anti- IgG antibody (1:1000, Beyotime, Shanghai, China) for 1 h at room temperature. Membranes were detected with HRP substrate luminol reagents (Merck-Millipore, Darmstadt, Germany), and scanned using the Gel Doc EZ Imager (Bio-Rad, Berkeley, CA, USA). Quantification of blot intensity was performed using ImageJ software (V1.8.0, NIH, MD).
5
Western Blot Analysis of AKT/mTOR Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMSCs were cultured in differentiating medium for 48 h. Cells (1×106) suspended in 0.5 ml of Laemmle buffer (50 mM tris pH6.8, 1.25% SDS, 10% glycerol) were lysed using SoniConvert sonicator (DocSense) and denatured by heating at 100°C for 15 min. Then, protein concentration was determined using BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (20 µg) was separated in each lane via 6–12% gradient SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes (EMD Millipore) and subsequently blocked with PBS containing 5% skimmed milk (Beyotime Institute of Biotechnology) at room temperature for 30 min. The membranes were incubated with primary antibodies against: AKT (1:3,000; cat. no. ab9905), AKT phosphor T308 (1:2,000; cat. no. ab38449), mTOR (1:1,000; cat. no. ab2732), mTOR phosphor S2448 (1:1,000; cat. no. ab109268) and β-actin (1:5,000; cat. no. ab8226; all from Abcam) for 1 h at room temperature. Membranes were washed three times with PBS containing 0.1% Tween-20 (Beyotime Institute of Biotechnology) and subsequently incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) for 1 h at room temperature. Proteins bands were visualized on X-ray film using enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.) and quantified using ImageJ software (version-2.0; National Institutes of Health).
6
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hPDLCs cultured in different exosomes were collected on day 7, 100 µL of RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology) was added, resuspended, lysed on ice, and then centrifuged for 20 min. The BCA method determined the extracted protein concentrations, which was adjusted by adding appropriate amounts of PBS and SDS‐PAGE protein buffer (Beyotime Biotechnology). The samples were boiled in a 97 °C water bath for 15 min and stored at −80 °C. A preformed gel plate was mounted, 5 µL of preset protein marker (Epizyme Biomedical Technology, Shanghai, China) was added as a reference for molecular protein weight, and samples were added for electrophoresis. After completion of electrophoresis, the film was removed and immersed in transfer solution for 5 min and transferred at a constant flow of 300 mA for 1 h. After removing the protein transfer NC film (Bio‐Rad, CA, USA), it was closed with 5% w/v skimmed milk (Beyotime Biotechnology) at room temperature 1 h. The primary antibody (Table S2 , Supporting Information) was incubated overnight at 4 °C on a shaker. The secondary antibody (Table S2 , Supporting Information) was incubated at room temperature for 1 h. Chemiluminescent substrates for HRP detection dipped in a biomolecular imager.
7
Western Blot Analysis of Kidney Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HK-2 cells and kidney tissues were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China), according to standard procedures. Then, the protein concentration was calculated using a BCA protein assay kit (Thermo Scientific, USA). Afterwards, the proteins were separated using SDS-polyacrylamide gel (8%-12%), transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corp., USA), and blocked by 5% skimmed milk (Beyotime, China) for 2 h. Next, the membranes were incubated with primary antibodies raised against the following: TOPK (Santa Cruz, USA); p-TOPK, NGAL (Abcam, USA); α-tubulin (Proteintech, USA); Aurora B, Bcl2, Bax, p-AKT and AKT (Cell Signaling Technology, USA).
8
Western Blot Analysis of CCT6A, CDC20, and GAPDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) 48 h post-transfection. The concentration of total protein was measured by BCA kit (Beyotime Institute of Biotechnology). The thermally denatured proteins (20 µg) were then separated by SDS-PAGE on a 4–20% precast gel (Beyotime Institute of Biotechnology). Subsequently, the proteins were transferred to a nitrocellulose membrane (Pall Corporation), which was incubated with CCT6A (cat. no. 19793-1-AP; 1:1,000; Wuhan Sanying Biotechnology), CDC20 (cat. no. 10252-1-AP; 1:10,000; Wuhan Sanying Biotechnology) and GAPDH (cat. no. AF7021; 1:2,000, Affinity Biosciences) antibodies at 4°C overnight after being blocked using 5% skimmed milk (Beyotime Institute of Biotechnology) at 37°C for 1.5 h. The membrane was successively incubated with a HRP-linked goat anti-rabbit secondary antibody (cat. no. S0001; 1:5,000; Affinity Biosciences) at 37°C for 1.5 h. An ECL kit (Beyotime Institute of Biotechnology) was adopted to visualize the protein bands. Finally, ImageJ (version 1.8; National Institutes of Health) was used to semi-quantify protein expression.
9
Western Blot for GPx-3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were crushed with liquid nitrogen and then lysed with Ripa lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Solarbio, Beijing, China). Total protein concentration was determined using a protein quantification kit (BCA kit) (Beyotime, Shanghai, China). Protein was separated by SDS-PAGE and transferred to the PVDF membrane (Millipore, America). The membranes were incubated with 5% skimmed milk (Beyotime, Shanghai, China) for 2 h at room temperature and then incubated with rabbit anti-human GPx-3 (1:1000, Abcam, UK) at 4 °C overnight. GAPDH (1:5000, Beyotime, Shanghai, China) was used as an internal reference. Finally, the membranes were incubated with the secondary antibody for 2 h at room temperature and visualized using an enhanced chemo luminescence kit (ECL, Beyotime, Shanghai, China).
10
Protein Expression Analysis Using SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This work employed RIPA buffer (Beyotime) for extracting total tissue and cell proteins. Thereafter, 12% SDS-PAGE (Beyotime) was performed to separate the extracted proteins, followed by transfer on PVDF membranes (Roche). Then, membranes were blocked using 5% skimmed milk (Beyotime) and incubated with primary antibodies (Abcam, Cambridge, MA, USA) overnight under 4°C, including anti-Ki67 (1 : 1000), anti-MMP9 (1 : 1000), and anti-GAPDH (1 : 5000). Afterwards, the secondary antibody HRP-labeled rabbit IgG (1 : 50,000, Abcam) was employed to further incubate bands. This work adopted enhanced chemiluminescence (ECL, Sangon, Shanghai, China) for protein visualization.
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!