The cells were treated with MAPK or ERK inhibitor for 24 h and 5×105 cells were collected for Western blot analysis. Protein (20 μg) was extracted from the treated cells, run on sodium-dodecyl sulfate-polyacrylamide gels (10%), and transferred to nitrocellulose membranes. The membranes were blocked by 5% non-fat milk for 2 h at room temperature. The following primary antibodies were used for overnight incubation at 4°C: anti-caspase-3 (1: 1000, bs-0081R, Bioss, Beijing, China), anti-P53 (1: 2000, Cell Signaling), anti-p-MAPK14 (1: 1000, bs-5476R, Bioss, Beijing, China), anti-MAPK14 (1: 1000, bs-28027R, Bioss, Beijing, China), anti-MEK1 (1: 1000, ABclonal), and anti-p-MEK1 (1: 1000, ABclonal). After washing, the membranes were incubated with the secondary antibody (1: 100, ab131368, Abcam) for 2 h at room temperature. An Enhanced Chemiluminescence kit (No. RPN2133; GE Healthcare Life Sciences, Chicago, IL, USA) was added to the membrane prior to visualization with a gel imaging system (Thermo Fisher Scientific).
Bs 5476r
Manufactured by Bioss Antibodies
Sourced in China, United States
The Bs-5476R is a laboratory equipment designed for general laboratory use. It serves as a centrifuge capable of separating liquid components based on their density differences. The core function of the Bs-5476R is to provide controlled and consistent centrifugal force to enable separation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0637r, by Bioss Antibodies (2 mentions)
Ab131368, by Abcam (1 mentions)
Gel imaging system, by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Bioss Antibodies (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Bs 0081r, by Bioss Antibodies (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Peroxidase conjugated affinipure goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ab85309, by Abcam (1 mentions)
Bs 0465r, by Bioss Antibodies (1 mentions)
Ac026, by ABclonal (1 mentions)
3 protocols using bs 5476r
1
MAP Kinase Pathway Protein Profiling
2
Profiling p38 MAPK in Lenok Gonad
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Proteins were extracted from gonad samples at 300 dph (immature, n = 3) and three years post-hatching (mature, n = 3) of female lenoks that were reared under identical conditions to individuals used in transcriptome analyses. To explore the expression profiles of p38 MAPK protein in different tissues, the proteins of the gill, heart, liver, spleen, intestine, skin, and ovary tissues were extracted from female lenok at 750 dph (n = 3), which was a time of rapid follicular development. The proteins were boiled for 20 min in SDS-PAGE loading buffer and separated using 12% SDS-PAGE gels. The proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad, USA) and analyzed by western blotting. Anti-p38 MAPK (bs-0637R, Bioss, 1:10000), anti-phospho-p38 MAPK (Thr180) (bs-5476R, Bioss, 1:5000), and beita-actin (ym3121, Immunoway, 1:5000) were used as primary antibodies, and peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:2000) was used as the secondary antibody (Cell Signaling Technology, USA). After washing with PBST, the protein bands were visualized by infrared fluorescence using the Odyssey Imaging System (LI-COR InC).
3
Protein Expression Analysis in Cells
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Total protein was extracted from the cells using RIPA lysis buffer, and the protein concentration was determined using the BCA assay kit. After denaturation at 95 °C, the protein was stored at -80 °C. Equal amounts of protein were loaded to each lane and separated by 10% SDS-PAGE. The separated proteins were then transferred onto PVDF membranes and blocked with 5% skim milk powder for 2 h. The PVDF membranes were then incubated overnight at 4 °C with appropriate primary antibodies against β-actin (AC026, Abclonal, 1:50,000), Sirt1 (60303-1-lg, Proteintech, 1:2000), AP-1 (66313-1-Ig, Proteintech, 1:2000), NF-κB p65 (bs-0465R, Bioss, 1:2000), NF-κB p-p65 (82335-1-RR, Proteintech, 1:2000), p-P38 (bs-5476R, Bioss, 1:2000), P38 (bs-0637R, Bioss, 1:2000), p-JNK (80024-1-RR, Proteintech, 1:2000), JNK (66210-1-lg, Proteintech, 1:2000), HO-1 (ab85309, Abcam, 1:3000), and Nrf2 (16396-1-AP, Proteintech, 1:2000). After washing with TBST, the membranes were incubated at room temperature for 2 h with Goat Anti-Rabbit IgG (H + L) HRP (S0001, Affbiotech, 1:5000), followed by detection using enhanced chemiluminescence (ECL). The bands were exposed using the fluorescence imaging system V2.0 (Tanon, Shanghai, China), scanned, and analyzed using Gel-Pro analyzer 4.0. The relative expression levels of the target proteins were calculated with β-actin as an internal reference.
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