Cells were homogenized in cold RIPA buffer with phenyl methane sulfonyl fluoride (PMSF) and phosphatase inhibitor. Protein concentrations were determined by the Bicinchoninic Acid (BCA) Protein Assay Kit (Sparkjade, Jinan, China). Equal protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, MA, United States). PVDF membranes were blocked with 5% skimmed milk powder and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study are as follows: DDIT4 (1:500; ER1706-76, HUABIO, Hangzhou, China), AKT1 (1:500; ER1609-47, HUABIO, Hangzhou, China), phosphorylated AKT1 (Ser473) (p-AKT1; 1:500; ER1607-73, HUABIO, Hangzhou, China), RP70S6KB1 (1:1,000; #2708, Cell Signaling Technology, Boston, United States), p-RP70S6KB1 (Thr389) (p-S6K; 1:1,000; #9234, Cell Signaling Technology, Boston, United States), Raptor (1:500; ER1802-57, HUABIO, Hangzhou, China), Rictor (1:500; EM1709-50, HUABIO, Hangzhou, China), and β-actin (1:1,000; R1207-1, HUABIO, Hangzhou, China). After staining with HRP-conjugated secondary antibody, protein bands were visualized using the Immobilon Western HRP Substrate Kit (Millipore, MA, United States).
Immobilon western hrp substrate kit
Manufactured by Merck Group
Sourced in United States
The Immobilon Western HRP Substrate Kit is a chemiluminescent substrate for the detection of horseradish peroxidase (HRP) in Western blotting applications. The kit provides the necessary components for the visualization of target proteins on a membrane after immunoblotting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
R1207 1, by Huabio (1 mentions)
Bca protein assay kit, by Sparkjade (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Smad4, by Abcam (1 mentions)
P smad2 3, by Abcam (1 mentions)
Hrp labeled secondary antibody, by Merck Group (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Antimouse igg conjugated horseradish peroxidase, by ZSGB-BIO (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Genegnome imaging system, by Syngene (1 mentions)
Amicon centrifugal filter device, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
8 protocols using immobilon western hrp substrate kit
1
Western Blot Analysis of mTOR Pathway
2
Western Blot and Immunoprecipitation Analysis
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [25 mM tris (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS] supplemented with protease inhibitor cocktail (Roche). The cell lysates were centrifuged at 12,000g for 15 min at 4°C and measured for protein concentrations using Bicinchoninic acid (BCA) protein assay reagent (Thermo Fisher Scientific). Equal amounts of protein were resolved by SDS-PAGE (polyacrylamide gel electrophoresis) using a standard protocol and transferred to a polyvinylidene difluoride membrane (0.45 mm). Primary and secondary antibodies were used at appropriate dilutions. Protein bands were visualized using the Immobilon Western HRP Substrate Kit (Millipore).
For immunoprecipitation analysis, total cell lysates were incubated with anti-Flag–conjugated beads or anti-hemagglutinin–conjugated magnetic beads overnight at 4°C. The pellets were washed for three times with IP buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40] and then resolved by SDS-PAGE.
For immunoprecipitation analysis, total cell lysates were incubated with anti-Flag–conjugated beads or anti-hemagglutinin–conjugated magnetic beads overnight at 4°C. The pellets were washed for three times with IP buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40] and then resolved by SDS-PAGE.
3
Quantitative Protein Expression Analysis
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Cells were routinely harvested as indicated time, and the irradiated cells were harvested at 24 h after radiation. Protein lysates were separated in SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in TBS buffer for 1 h, and then incubated with primary antibodies overnight at 4 °C. At room temperature, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA) for 2 h. The reaction was visualized using Immobilon™ Western HRP substrate kit (Millipore, USA). The primary antibodies were as follows: TGF-β2 and c-Myc (Santa Cruz, USA), TGF-βRIII (CST, USA), ARHGEF15 (Abcam, USA), ABL2 (Abcam, USA), p-SMAD2/3 (Abcam, USA), E2F6 (Abcam, USA), SMAD4 (Abcam, USA), GAPDH (CST, USA). SMAD4 protein expression in pancreatic cells was tested by western blot (Additional file 4 : Figure S3B).
4
Western Blot Analysis of AIFM1 Mutants
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Total protein was extracted from the HEK293 cells transfected with the WT and mutant AIFM1 plasmids. The proteins were denatured and separated by 10% SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Merck Millipore, China). After blocking, the membranes were incubated with mouse monoclonal anti-GFP antibody (Proteintech, Wuhan, China) and mouse monoclonal anti-β-actin antibody (ZSGB-Bio, Beijing, China), and followed by anti-mouse IgG conjugated horseradish peroxidase (ZSGB-Bio, Beijing, China). Finally, the immunoblots were detected with an Immobilon Western HRP Substrate kit (WBKLS0100, Millipore, Schaffhausen, Switzerland) using the enhanced chemiluminescence system (Tanon5200, Shanghai, China). The gray scale of each band was quantified with ImageJ software and normalized by β-actin.
5
Western Blot Protein Quantification
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The protein concentrations of whole-cell lysates were quantified using DC™ Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, 35 µg of proteins were resolved by denaturing SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes overnight. Membranes were blocked with 5% non-fat dried milk in TBST for 1 h at room temperature (RT) and incubated with primary antibodies at 4 °C overnight. After washing the membranes three times with TBST for 5 min they were incubated with HRP-conjugated secondary antibodies for 1 h at RT. Finally, the membranes were washed again three times with TBST, and signals were detected using the Immobilon Western HRP substrate kit (WBKLS0500, Merck Millipore, Burlington, MA, USA) with the GeneGnome imaging system (Syngene, Bengaluru, India).
6
Western Blot Protein Detection Protocol
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The conditioned media derived from 12-well plates were concentrated approximately five times using Amicon centrifugal filter device (Millipore, Danvers, MA, USA; 3 kDa MWCO), and then used for Western blot analysis as previously described [27 (link)]. An equal amount of each sample was resolved on an SDS-polyacrylamide gel and electroblotted onto a PVDF membrane. The blots were blocked with 5% skim milk in PBS containing 0.05% Tween 20 and incubated with an appropriate antibody (diluted 1:1000 in blocking solution). Immobilon Western HRP Substrate kit (Merck, Kenilworth, NJ, USA) and ImageQuant LAS 4000 software (GE Healthcare, Chicago, IL, USA) were used to detect Chemiluminescence signals. Band intensities were quantified using ImageJ software. One representative Western blot for each experiment is shown in the figures. Data from three independent experiments were averaged for each condition and presented in the form of bar graphs. Data are expressed in fold changes compared with control (mean ± SD). Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA), and p-values were calculated by paired t-test.
7
Western Blot Analysis of Gemcitabine Resistance
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RIPA lysis buffer (Thermo Fisher Scientific, Inc.) was used to lyse and extract total protein from PANC-1 gemcitabine-resistant and parent cell lines. Protein concentration was determined used a BCA protein assay and protein lysates (25 ng/µl; 6 µl/lane) were separated via 10 or 12% SDS-PAGE according to molecular weight. Subsequently, separated protein bands were transferred onto polyvinylidene fluoride membranes (PVDF) (EMD Millipore) and blocked with 5% skimmed milk for 1 h at room temperature. Next, the membranes were first probed with the relevant primary antibodies overnight at 4°C and subsequently incubated with HRP-labeled secondary antibodies for 2 h at room temperature for visualization using Immobilon™ Western HRP Substrate kit (EMD Millipore). The antibodies used are listed in Table SIII .
8
Kaempferol Modulates Intracellular Calcium and Mitochondrial Membrane Potential
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HUVECs were treated with 100 µM kaempferol for 6, 12 and 24 h. Cells were then harvested and labeled with 2 µM Fluo-4/AM (a specific intracellular Ca 2+ fluorescence probe) and 500 nM DiOC 6 (3) , respectively, at 37˚C for 30 min. Consequently, intracellular Ca 2+ and ∆Ψm were individually analyzed for fluorescence intensity by flow cytometry as previously described (17) .
Western blot analysis. HUVECs (5x10 6 cells) were incubated in 100 µM kaempferol for 0, 12 or 24 h. After being harvested and lysed, the 10% SDS-polyacrylamide electrophoresis (SDS-PAGE) gels were used to separate equal amount of protein extract from cell lysate as detailed by Yang et al (18) . The appropriate the primary antibodies were hybridized to observe the specific protein signals. Then the HRP-conjugated secondary antibodies were applied before using Immobilon Western HRP substrate kit (Merck Millipore). The densitometric quantification of each band was performed utilizing NIH ImageJ 1.47 software.
Western blot analysis. HUVECs (5x10 6 cells) were incubated in 100 µM kaempferol for 0, 12 or 24 h. After being harvested and lysed, the 10% SDS-polyacrylamide electrophoresis (SDS-PAGE) gels were used to separate equal amount of protein extract from cell lysate as detailed by Yang et al (18) . The appropriate the primary antibodies were hybridized to observe the specific protein signals. Then the HRP-conjugated secondary antibodies were applied before using Immobilon Western HRP substrate kit (Merck Millipore). The densitometric quantification of each band was performed utilizing NIH ImageJ 1.47 software.
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