Total protein was extracted from the liver of experimental fish with Total Protein Extraction Kit (Applygen Technologies Inc, Beijing, China), and nuclear and cytosolic fractions were collected using a nuclear protein extraction kit (Pierce, Nashville, TN, USA), according to the instructions of the manufacturers. Protein concentration was measured using BCA kit (Pierce). Fifty micrograms of protein was loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). The membranes were incubated with 5% skimmed milk for 1 h at room temperature. Immunoblots were obtained using antibodies against the following proteins: ERK1/2, ERK1/2 (pThr202/Tyr204), JNK1/2, JNK1/2(pThr183/Thr185), p38, p38 (pThr180/Thr182), IKKα/β, IKKα/β (pSer176/Ser180), IκBα, IκBα (pSer32/Ser36), c-Jun, c-Jun (pSer73), NF-κB p65 and beta-actin (Cell Signaling Technology, Danvers, MA, USA). Western blots were exposed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) and film images were scanned by Epson Perfection V33 (China).
Polyvinylidene difluoride membrane
Manufactured by Pall Corporation
Sourced in United States
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory equipment used for various applications. PVDF is a thermoplastic fluoropolymer material with high chemical resistance and mechanical strength. These membranes are designed for filtration, separation, and analysis purposes in scientific research and industrial settings.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ecl western blotting kit, by Cytiva (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Immune precipitation assay buffer, by Keygen Biotech (3 mentions)
Bca protein assay kit, by Keygen Biotech (3 mentions)
Odyssey machine, by LI COR (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Chemidoc xrs plus, by Bio-Rad (2 mentions)
Chemi lumi one l, by Nacalai Tesque (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Bca protein quantification kit, by Vazyme (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence kit, by Beyotime (2 mentions)
Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (2 mentions)
Ecl western blotting kit, by Advansta (2 mentions)
Phenylmethylsulfonyl fluoride, by Keygen Biotech (2 mentions)
Phosphatase inhibitor cocktail, by Keygen Biotech (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Hrp conjugated secondary antibody, by Beyotime (2 mentions)
64 protocols using polyvinylidene difluoride membrane
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Cellular Signaling Proteins
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Following treatment, cells were harvested in the radioimmunoprecipitation assay buffer. Equal amounts (20 μg protein) of cellular lysates were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The isolated proteins were then transferred by electroblotting to polyvinylidene difluoride membranes (Pall Corporation, Washington, NY, USA), then subsequently incubated with the epitope-specific primary antibodies diluted 1:1000 (Cell Signaling, Boston, MA, USA) against the following proteins: phospho-extracellular signal-regulated kinase (P-ERK, # 9101S), ERK (# 4695S), phospho-Akt (P-Akr, # 4060S), Akt (# 4691S), phospho-phosphatidylinositol 3-kinase (P-PI3K, # 17366S), PI3K (# 4249S), Phospho-mammalian target of rapamycin (P-mTOR, # 5536S), mTOR (# 2983S), Phospho-vascular endothelial growth factor receptor 2 (VEGFR2, # 2478S), VEGFR2 (# 9698S), matrix metalloproteinase-9 (MMP-9, # 13667S), matrix metalloproteinase-2 (MMP-2, # 40994S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, # 5174S). A horseradish peroxidase-conjugated rabbit antibody diluted 1:2000 (Cell Signaling) was used as a secondary antibody.
3
Protein Expression Analysis in HCC
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Total protein is isolated from cultured cells, HCC tissues and adjacent healthy tissues, as well as formed tumors in nude mice utilizing the protein extraction kit (Solarbio) and quantified using bicinchoninic acid assay Kit (Beyotime). After separation through sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE), protein samples are subjected to transfer onto polyvinylidene difluoride membranes (Pall Corporation, East Hills, NY, USA). Later, the membranes are blocked in defatted milk, incubated with special primary antibody against Raf1 (ab173539, 1:1500), snail (ab216347, 1:1000), E-cadherin (ab11512, 1:1000), MEK1 (ab32091, 1:1000), phosphorylated MEK1 (p-MEK1; ab96379, 1:1500), ERKs (ab218017, 1:1000), phosphorylated ERKs (p-ERKs; ab223500, 1:1000), or GAPDH (internal control; ab181602, 1:2000), then incubated with horseradish peroxidase-conjugated secondary antibody (ab205718, 1:5000). Visualization of protein blots is achieved using the enhanced chemiluminescence kit (Beyotime).
4
Protein Extraction and Analysis by Western Blot
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Total protein was extracted by RIPA lysis buffer containing protease inhibitors and was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall Corporation, Mexico, USA). The membranes were blocked in StartingBlock (Genscript ProBio, Nanjing, China) and stained with the appropriate primary and secondary antibodies. After washing with PBST (phosphate-buffered saline with Tween), the membranes were scanned and analyzed by ODYSSEY machine (LI-COR, American). The antibodies used are listed in Supplementary Table 4 .
5
Western Blot Analysis of Protein Extracts
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Cell pellets were lysed for 30 min at 4 °C in a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 350 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 10% glycerol, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride and phosphatase inhibitor cocktails. Whole-cell extracts were resolved on SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Pall Corporation, Pensacola, FL, USA). The membranes were blocked in a blocking buffer and incubated with specific primary antibodies for the target molecules at 4 °C overnight. The signals were detected using an ECL detection kit (iNtRON Biotechnology, Daejeon, Korea), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.
6
Western Blot Analysis of EMT Markers
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Cell and tumor lysates were subjected to electrophoresis on 8–10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). Then, the membranes were incubated with primary antibodies against USP47, vimentin, N-cadherin, Snail, Sox9, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (BD Bioscience, San Jose, CA, USA), or ubiquitin (Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein blots were visualized with an enhanced chemiluminescence detection kit.
7
Western Blot Analysis of Signaling Proteins
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Each fraction of proteins was prepared in Laemmli sample buffer (Sigma-Aldrich) and heated in boiling water for 5 minutes. All proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA), and blocked (5% nonfat milk in Tris-buffered saline with 0.1% Tween 20) for 60 minutes at room temperature. The membranes blotted with total proteins were then exposed separately to rabbit monoclonal primary antibodies against β-actin (1:1,000, cell signaling technology [CST]), p38 (1:1,000, CST), phospho-p38 (1:1,000, CST), ERK1/2 (1:1,000, CST), phospho-ERK1/2 (1:1,000, CST), JNK (1:1,000, CST), phospho-JNK (1:1,000, CST); phospho-IκBα (1:1,000, CST), and IκBα (1:1,000, CST). The membranes blotted with cytoplasmic proteins were exposed to rabbit monoclonal primary antibodies against NFκB (p65) (1:500, CST) and β-actin (1:1,000, CST), whereas the membranes blotted with nuclear proteins were exposed to rabbit monoclonal primary antibodies against NFκB (p65) (1:500, CST) and lamin B (1:500, Abcam). IRDye 800CW goat anti-rabbit IgG (H+L) (Li-Cor Biosciences, Lincoln, NE, USA) was used as the secondary antibody. The signal intensities of the bands were analyzed using the Odyssey infrared imaging system (Li-Cor).
8
Western Blot Protein Analysis Protocol
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Cells were washed with cold PBS at 4°C and directly lysed in Laemmli sample buffer (62.5 mM Tris [pH 6.8]; 2% sodium dodecyl sulfate [SDS]; 20% glycerol). Cell lysates were sonicated with a Branson Sonifier 150 (Branson Ultrasonics) at setting 4 with three 10‐s pulses. After determining the protein concentration of each sample, 2‐mercaptoethanol and bromophenol blue were added to the lysates at concentrations of 5% and 0.025% (v/v), respectively. The lysates were boiled for 7 min and used as whole cell lysates. Equal amounts of protein (3–7 μg) were subjected to electrophoresis using 10% or 4%–20% gradient polyacrylamide gels. The electrophoretically separated proteins were transferred to polyvinylidene difluoride membranes (Pall Corporation) using a submarine transfer apparatus (Criterion Blotter, Bio‐Rad). Immunoblotting was performed using standard procedures.29
9
Western Blotting Protocol for Protein Analysis
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Western blotting was performed as described previously [52 (link)]. Briefly, cells were lysed in SDS sample buffer containing 2 µg/mL aprotinin (Seikagaku Kogyo, Tokyo, Japan), 0.8 µg/mL pepstain A (Wako Pure Chemicals, Osaka, Japan), 2 µg/mL leupeptin (Nacalai Tesque), 2 mM PMSF (Nacalai Tesque), 20 mM β-glycerophosphate (MilliporeSigma), 50 mM NaF (Wako), and 10 mM Na3VO4 (Wako) and denatured at 40 and 100 °C for 20 and 5 min, respectively. Cell lysate components were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with Blocking One (Nacalai Tesque) and incubated for 1 h at room temperature or overnight at 4 °C with primary and secondary antibodies diluted in Tris-buffered saline containing 5% Blocking One and 0.1% Tween20. Sequential reprobing of the membranes with various antibodies was performed after the inactivation of HRP by 0.1% NaN3. Proteins were detected with Chemi-Lumi One L (07880-70, Nacalai Tesque) and Clarity (#1705061, Bio-Rad, Hercules, CA, USA) using an image analyzer ChemiDoc XRSplus (Bio-Rad). Full-length blots of Figure 1 , Figure 3 , Figure 6 , and Figure S2 are shown in Figure S5–S8 .
10
Quantifying SATB1 Protein Levels
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Cells and fresh tissues were lysed in 1×sodium dodecyl sulfate (SDS) lysis buffer. Equal amounts of total protein were separated by SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Pall Corporation, Mexico City, Mexico), and incubated overnight at 4°C with a SATB1 rabbit polyclonal antibody (1∶500 dilution, PRS4631; Sigma-Aldrich, St. Louis, MO, USA). Beta-actin (1∶10000, Sigma-Aldrich) was used as a loading control. Protein bands were evaluated by enhanced chemiluminescence (Thermo Fisher Scientific, RockFord, IL, USA).
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