The uterus was sectioned, and slices were incubated and lysed in RIPA lysis buffer (C1053; Applygen Technologies, Beijing, China) supplemented with protease inhibitor (P1265; Applygen Technologies). The protein concentration was quantified with bicinchoninic acid (P1511; Applygen Technologies). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using a 10% polyacrylamide gel, and the samples were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blotted with anti-LIF (sc-1336; Santa Cruz Biotechnology, Heidelberg, Germany) or anti-Ang-2 (AP23297PU-N; Acris Antibodies, Herford, Germany) primary antibodies at a 1:1,000 dilution and incubated overnight at 4°C. Following incubation, the membranes were washed 3 times with Tris-buffered saline and Tween 20 buffer and then incubated with the secondary antibodies (P1308, P1309; Applygen Technologies) at a 1:10,000 dilution at room temperature for 1 h. The blots were visualized using the Super ECL Plus detection reagent (P1010; Applygen Technologies). The enhanced chemiluminescence signals were detected using Quantity One software (Bio-Rad). GAPDH (blotted with ab8245; Abcam, Cambridge, UK) was used as an internal control to validate the quantity of protein loaded onto the gel.
P1265
Manufactured by Applygen
Sourced in China
P1265 is a multi-purpose lab equipment designed for general laboratory applications. It features a compact and durable construction, with precise controls for consistent performance. The core function of P1265 is to assist in various laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Super ecl plus detection reagent, by Applygen (2 mentions)
P1260, by Applygen (2 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
P1308, by Applygen (1 mentions)
Ab8245, by Abcam (1 mentions)
P1511, by Applygen (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti hsp90, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho ampkα, by Cell Signaling Technology (1 mentions)
Rabbit anti ampkα, by Cell Signaling Technology (1 mentions)
Rabbit phospho erk, by Cell Signaling Technology (1 mentions)
C1053, by Applygen (1 mentions)
Goat anti rabbit igg hrp, by ZSGB-BIO (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
No 22915 1 ap, by Proteintech (1 mentions)
Ab8448, by Abcam (1 mentions)
C1309, by Applygen (1 mentions)
4 protocols using p1265
1
Western Blot Analysis of Uterine Proteins
2
Effects of SZ-A, FAG, and DAB on Islets and MIN6 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The islets of KKAy mice were incubated in RPMI-1640 medium containing vehicle or 100 μg/ml SZ-A for 24 h. MIN6 cells were fasted for 1 h in Krebs buffer (2.8 mM glucose) with or without different concentrations of SZ-A, FAG, or DAB. Then, they were cultured for another 1 h in new Krebs buffer containing 2.8 mM or 16.8 mM glucose combined with SZ-A, FAG, DAB or vehicle. High glucose- and palmitic acid-treated MIN6 cells were coincubated with different concentrations of SZ-A, DNJ or vehicle for 24 h.
Islets or MIN6 cells were homogenized in RIPA lysis buffer (C1053; APPLYGEN, China) containing protease inhibitors (P1265; APPLYGEN, China) and phosphatase inhibitors (P1260; APPLYGEN, China). Protein (10 μg) from each sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for immunoblotting. Rabbit anti-phospho-AMPKα (1:1000, 2535s), rabbit anti-AMPKα (1:1000, 2532s), rabbit-phospho-Erk (1:1000, 9101s), rabbit anti-Erk (1:1000, 9102s), and rabbit anti-HSP90 (1:1000, 4877s) were purchased from Cell Signaling Technology (USA). Goat anti-rabbit IgG/HRP (1:5000, ZDR-5306) was purchased from ZSGB-BIO (China).
Islets or MIN6 cells were homogenized in RIPA lysis buffer (C1053; APPLYGEN, China) containing protease inhibitors (P1265; APPLYGEN, China) and phosphatase inhibitors (P1260; APPLYGEN, China). Protein (10 μg) from each sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for immunoblotting. Rabbit anti-phospho-AMPKα (1:1000, 2535s), rabbit anti-AMPKα (1:1000, 2532s), rabbit-phospho-Erk (1:1000, 9101s), rabbit anti-Erk (1:1000, 9102s), and rabbit anti-HSP90 (1:1000, 4877s) were purchased from Cell Signaling Technology (USA). Goat anti-rabbit IgG/HRP (1:5000, ZDR-5306) was purchased from ZSGB-BIO (China).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In brief, tissue proteins were extracted in RIPA lysis buffer (C1053; Applygen Technologies Inc.; Beijing, China) containing protease inhibitor (P1265; Applygen Technologies Inc.; Beijing, China) and protein phosphatase inhibitor mixture (P1260; Applygen Technologies Inc.; Beijing, China). The quantification of proteins was measured by BCA protein assay kit (Beyotime Biotechnology Inc.; Shanghai, China). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA) then incubated overnight at 4°C with antibodies against GSDMD (abcam, No.ab219800, 1:1000), caspase-1 (Proteintech, No.22915-1-AP, 1:500), and GAPDH (Proteintech, No.60004-1-lg, 1:5000). The goat-anti-rabbit IgG-HRP (Proteintech, No.SA00001-2, 1:10000) secondary antibody was used. Enhanced chemiluminescence was used for color development. The gray values of the protein bands were analyzed by Image J, and the relative protein expression was calculated using GAPDH as the internal standard.
4
Western Blot Analysis of Angiogenesis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples of the upper genital tract were homogenized and lysed in ice-cold RIPA lysis buffer (C1053+; Applygen Technologies, Beijing, China) supplemented with protease inhibitor (P1265; Applygen Technologies). The proteins were in the supernatants after the homogenates were centrifuged at 12000 ×g for 20 min at 4°C. The proteins were quantified with bicinchoninic acid (BCA) (P1511; Applygen Technologies) and resolved in SDS-PAGE loading buffer at 95°C for 10 min. After loading (20 μg protein), proteins were separated in 10% Tris-Bis gel and then transferred to nitrocellulose membranes by electroblotting. After blotting in 5% of nonfat dry milk in TBST for 30 min at room temperature, the membranes were incubated with anti-VEGF (1 : 1000, ab46154; Abcam, Cambridge, UK) or anti-Ang-2 (1 : 1000, SC-20718; Santa Cruz Biotechnology, Heidelberg, Germany) or anti-OPN (1 : 1000, ab8448; Abcam, Cambridge, UK) antibodies overnight at 4°C, followed by incubation with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1 : 5000, C1309; Applygen Technologies) for 1 hour at room temperature. The blots were visualized using the Super ECL Plus detection reagent (P1010; Applygen Technologies). The enhanced chemiluminescence signals were detected using Quantity One software (Bio Rad).
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!