A375 and MV3 melanoma cell lines were harvested, then suspended in RIPA Lysis Buffer. Protein concentrations were detected with BCA protein assay kit (Beyotime Biotech, China). The lysates from cells as well as the fresh tissues were separated by SDS-PAGE, followed by transferring onto PVDF membranes (Millipore, USA). Followed by blocking with 5% BSA, the PVDF membranes were incubated gently with a primary antibody against human GAPDH (1:1000, Beyotime), p21 (1:1000, Cell Signaling), CDK2 (1:1000, Cell Signaling), Cyclin E (1:1000, Cell Signaling), E-cadherin (1:1000, Cell Signaling) and vimentin (1:1000, Cell Signaling) at 4°C overnight, followed by appropriate (horseradish peroxidase-conjugated secondary antibody) HRP-conjugated secondary antibodies. HRP-labeled goat anti-mouse IgG (H + L) (A0216, 1:2000) and goat anti-rabbit IgG (H + L) (A0208 1:2000) were used as secondary antibodies which purchased from Beyotime. Proteins were visualized by ECL Western blot analysis system. The signal was captured by the ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).
Western blotting detection instruments
Manufactured by Clinx
Sourced in China
Western blotting detection instruments are laboratory equipment used to identify and quantify specific proteins within a complex mixture. They facilitate the detection and analysis of proteins separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Hrp labeled goat anti mouse igg h l, by Beyotime (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg h l, by Beyotime (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Cyclin e1, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Ab156034, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Mouse anti taz, by BD (1 mentions)
Mouse anti yap sc 101199, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh, by Beyotime (1 mentions)
Cell cycle regulation antibody sampler kit 9932, by Cell Signaling Technology (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Vimentin, by Proteintech (1 mentions)
Ecl system, by Beyotime (1 mentions)
6 protocols using western blotting detection instruments
1
Profiling Melanoma Cell Signaling
2
Morusin Concentration Effects on Cell Signaling
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After treated with different concentration gradient or time gradient of morusin (DMSO was used as control), we harvested all group cells and lysed with RIPA lysis buffer (Abcam, ab156034), then centrifuged (10000 g, 15 min) and separated the supernatants. 10% SDS-PAGE was used to separate lysated proteins and transferred to PVDF membrane. PVDF membrane containing total proteins was blocking with 5% defatted milk for 2 h, then incubated with primary antibody against C-myc (1:1000 Abcam ab32072), CDK1 (1:2000 Abcam ab18), CDK2 (1:1000, Cell Signaling Technology 9932T), CDK4 (1:1000, Cell Signaling Technology 9932T), CDK6 (1:1000, Cell Signaling Technology 13331T), Cyclin A1 (1:1000, Abcam ab53699), Cyclin B1 (1:1000, Cell Signaling Technology 12231T), Cyclin D1 (1:1000, Cell Signaling Technology 9932T), Cyclin E1 (1:1000, Cell Signaling Technology 9932T), Tubulin (1:2000, Enogene E12-043-03) at 4°C overnight. After washing with PBS for 3 times, PVDF membrane were incubated with secondary antibody HRP-labeled goat anti-mouse IgG (H+L) (A0216, 1:10000) or goat anti-rabbit IgG (H+L) (1:10000, A0208) at room temperature for 2 h. The signal was captured by ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).
3
Profiling Neuroblastoma Protein Signatures
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Proteins were extracted from neuroblastomas cells, and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF). After blocked with 5% nonfat milk in TBST for 2 hours, the membrane was incubated with primary antibodies. Membranes were washed three times and incubated with the horseradish peroxidase-conjugated second antibodies. The signals were captured by the ECL reagent (Beyotime) and visualized by western blotting detection instruments (Clinx Science). Mouse anti-TAZ (560235; BD Biosciences), mouse anti-YAP (sc-101199, 1:200), goat anti-CTGF (sc-14939, 1:200) from Santa Cruz company, rabbit anti-PDGF-β (E1A0240-1, 1:1000) from EnoGene, and mouse anti-GAPDH (AG019, 1:1000) from Beyotime, cell cycle regulation antibody sampler kit #9932 from Cell Signaling Technology were used as primary antibodies. HRP-labeled goat anti-mouse IgG (H + L) (A0216, 1:5000) and goat anti-rabbit IgG (H + L) (A0208) were used as secondary antibodies which purchased from Beyotime.
4
Western Blot Assay Protocol Detailed
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The Western blot assay was conducted as previously described.26 (link) All the primary antibodies used in this study were purchased from the Cell Signaling Technology (CST, Danvers, MA, USA) and are listed here: GAPDH (51132), CDK6 (3136), cyclin D1 (2978), E-cadherin (3195), vimentin (5741), β-catenin (8480), MEK1/2 (4694), p-MEK1/2 (Ser221, 2338), ERK1/2 (4695) or p-ERK1/2 (Thr202/Tyr204, 4370). HRP (horseradish peroxidase)-conjugated secondary antibodies including goat anti-mouse IgG (H+L) (A0216, 1:2,000) and goat anti-rabbit IgG (H+L) (A0208 1:2,000) were purchased from Beyotime (Taicang, Jiangsu, China). Proteins were visualized by the BeyoECL Plus (Beyotime) and Western blotting detection instruments (Clinx Science, Shanghai, China). The gray levels of the protein bands were calculated by using Image J 1.8.0 software (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/ ), and the relative protein levels were normalized by grey levels of GAPDH. Each blot was analyzed for 3 times, and a two-tailed unpaired Student’s t-test was performed to analyze the significance.
5
Molecular Profiling of Cervical Cancer Cells
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After being treated with DMSO or PD at indicated concentrations, human cervical cancer cells were harvested, and the Western blot assay was performed as previously described (Zhang et al., 2018 (link)). All the primary antibodies used in this study are listed here: p21 (1:1,000, Cell Signaling Technology, United States), p27 (1:1,000, Cell Signaling Technology, United States), CDK2 (1:1,000, Cell Signaling Technology, United States), CDK4 (1:1,000, Cell Signaling Technology, United States), Cyclin D1 (1:1,000, Cell Signaling Technology, United States), Cyclin E1 (1:1,000, Cell Signaling Technology, United States), E-cadherin (1:1,000, Cell Signaling Technology, United States), N-cadherin (1:1,000, Cell Signaling Technology, United States), Snail (1:1,000, Cell Signaling Technology, United States), Slug (1:1,000, Proteintech, China), Vimentin (1:1,000, Proteintech, China), Tubulin (1:1,000, Cell Signaling Technology, China), and c-Myc (1:1,000, Abcam, United States). horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H + L) (A0216, 1:1,0000) or goat anti-rabbit IgG (H + L) (A0208, 1:1,0000) are used as a secondary antibody. Proteins were finally visualized by the electro-chemiluminescence (ECL) system (Beyotime, China) and then captured by Western blotting detection instruments (Clinx Science).
6
Leflunomide-Induced Apoptosis Pathway Analysis
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Cells were treated with leflunomide for 72 h, then cells were collected, after that proteins were extracted by RIPA Lysis Buffer which contained 1% Phenyl methane sulfonyl fluoride (PMSF), stockpiled at −80°C. The proteins were separated by SDS-PAGE, subsequently, transfered onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% BSA for 2 h, and incubated with a primary antibody against human Tubulin (1:1000, Beyotime), LC3B (1:1000, Cell Signaling), CDK2 (1:1000, Cell Signaling), CyclinA2 (1:1000, Cell Signaling), DHODH (1:1000, Cell Signaling), BCL-2 (1:1000, Cell Signaling), Caspase-9 (1:1000, Cell Signaling), Caspase-3 (1:1000, Cell Signaling), pT172-AMPK (1:800, Cell Signaling), AMPK (1:800, Cell Signaling), pSer555-Ulk (1:1000, Cell Signaling), Ulk (1:1000, Cell Signaling), pThr 183/186-JNK (1:2000, Abcam), JNK (1:1000, Abcam), BCL-2 (1:1000, Cell Signaling) and Beclin1 (1:1000, Cell Signaling) at 4°C overnight. Then incubated with homologous secondary antibodies HRP-labeled goat anti-rabbit IgG (H+L) (1:2000, Beyotime) or goat anti-mouse IgG (H+L) (1:2000, Beyotime) for 2 h. Finally, the signal was captured by the ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).
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