PLA was bought from Sigma-Aldrich (St. Louis, MO, USA). And PD98059, an inhibitor of ERK1/2, was also taken from Sigma-Aldrich. PD98059 was dissolved in dimethyl sulfoxide which was purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was from Gibco (Australian origin). Anti-ERK1/2 and anti-phospho-ERK1/2 were from ImmunoWay Biotechnology (Plano, TX, USA). Anti-Bax, anti-Bcl-2, and anti-caspase-3 were from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin was acquired from Zhongshanjinqiao Biotechnology (Beijing, China). Annexin V-FITC Apoptosis Detection Kit with propidium iodide (PI) double staining flow cytometry was purchased from Bestbio Biotechnology Company (Shanghai, China).
Anti erk1 2
Manufactured by Immunoway
Sourced in United States
Anti-ERK1/2 is a lab equipment product that specifically targets and binds to the extracellular signal-regulated kinases 1 and 2 (ERK1/2). It is used in research applications to study the role of ERK1/2 in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti p erk1 2, by Immunoway (2 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Pd98059, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin, by Zhongshan Jinqiao Biotechnology (1 mentions)
Cell lysate, by Beyotime (1 mentions)
Anti gapdh, by Immunoway (1 mentions)
Anti akt, by Immunoway (1 mentions)
Anti p akt, by Immunoway (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Anti runx2, by Abcam (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti alp, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
4 protocols using anti erk1 2
1
Apoptosis Regulation by ERK1/2 Inhibition
2
Protein Expression Analysis Protocol
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The total protein was prepared using cell lysates (Beyotime, Shanghai, China), and the concentration of protein was determined with the BCA protein assay kit (Thermo, Massachusetts, USA). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose filter membrane. Membranes were treated with 5 % skimmed milk powder (Biolab, Beijing, China) and incubated with primary antibodies at 4 °C overnight, and then secondary antibodies (Immunoway, Suzhou, China) at room temperature for 1.5 h. The following antibodies were purchased: anti-FGF6 (Immunoway, SuZhou, China), anti-GAPDH (Immunoway, SuZhou, China), anti-AKT (Immunoway, SuZhou, China), anti-P-AKT (Immunoway, SuZhou, China), anti-ERK1/2 (Immunoway, SuZhou, China), anti-P-ERK1/2 (Immunoway, SuZhou, China), anti-FGFR (Immunoway, SuZhou, China), and anti-P-FGFR (Immunoway, SuZhou, China). After an appropriate amount of 1:10,000 diluted Secondary antibody incubated, ECL reagent (FuDeBio, Hangzhou, China) was used to detect the bands, and Quantity One (Bio-Rad, Hercules, CA) was used to analyze the positive bands.
3
Western Blot Analysis of Protein Signaling
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The four groups of cells grown in 60 mm dishes were cultured according to corresponding medium for 24 hours or 48 hours. Then, they were lysed using Radio-Immunoprecipitation assay (RIPA) lysis buffer from Thermo Fisher Scientific (Waltham, MA, USA) at 4 °C for 30 minutes, and subjected to 12000 rpm centrifugation at 4 °C for 5 minutes. The protein concentration was measured by BCA protein assay Kit from Thermo Fisher Scientific (Waltham, MA, USA). The lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk. The blots were analyzed with antibodies according to the manufacturer's instructions and visualized by peroxidase and an enhanced chemiluminescence system from Pierce Biotechnology (Waltham, MA, USA). The corresponding antibodies were used in the present study including anti-p-ERK1/2 and anti-ERK1/2 (Cat No: YP1197, Immuno Way, USA; Cat No: ab65142, Abcam, Cambridge, UK, respectively); anti-PKCα and anti-β-actin (Cat No: ab32376 and ab8227, Abcam, Cambridge, UK, respectively). Goat anti-rabbit IgG secondary antibody (Cat No: ab205718, Abcam, Cambridge, UK).
4
Investigating Cell Signaling Pathways
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The
MC3T3-E1 cell samples would be treated
in a similar manner to the alkaline phosphatase activity assay or
added to PD98059 for inhibition of the MEK enzyme site. The protein
in the sample collected by the cell scraper would be extracted by
the total protein extraction kit, which would be separated by electrophoresis
on polyacrylamide gel. Later, the separated protein would be transferred
to the PVDF membrane and then blocked with skimmed milk. After incubation
with the primary antibody (anti-ERK1/2, Immunoway, USA; anti-Perk1/2,
Cell Signaling Technology, USA; anti-OC, ABclonal, USA; anti-ALP,
Abcam, USA; anti-RUNX2, Abcam, USA; and anti-GAPDH, Abcam, USA) and
the secondary antibody (anti-Rabbit, Immunoway, USA), imaging was
taken under the action of the developer.
MC3T3-E1 cell samples would be treated
in a similar manner to the alkaline phosphatase activity assay or
added to PD98059 for inhibition of the MEK enzyme site. The protein
in the sample collected by the cell scraper would be extracted by
the total protein extraction kit, which would be separated by electrophoresis
on polyacrylamide gel. Later, the separated protein would be transferred
to the PVDF membrane and then blocked with skimmed milk. After incubation
with the primary antibody (anti-ERK1/2, Immunoway, USA; anti-Perk1/2,
Cell Signaling Technology, USA; anti-OC, ABclonal, USA; anti-ALP,
Abcam, USA; anti-RUNX2, Abcam, USA; and anti-GAPDH, Abcam, USA) and
the secondary antibody (anti-Rabbit, Immunoway, USA), imaging was
taken under the action of the developer.
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