Whole cell protein extracts were resolved by SDS-PAGE and proteins were transferred to nitrocellulose membranes. After blocking for 1hr at room temperature in 5% milk in PBS/0.1% Tween-20, membranes were incubated overnight at 4°C with the indicated primary antibodies. ABCB1 antibody (SC-8313, rabbit-polyclonal, 1:500 dilution) was purchased from Santa Cruz Biotechnology. PARP antibody (9542, rabbit-polyclonal antibody, 1:1000 dilution) was purchased from Cell Signaling Technology. Cleaved-PARP antibody (9541, rabbit-polyclonal antibody, 1:1000 dilution) was purchased from Cell Signaling Technology. Tubulin (T5168, mouse monoclonal antibody, 1:6000 dilution) was purchased from Sigma-Aldrich. Tubulin was used to monitor the amounts of samples applied. Following secondary antibody incubation, proteins were visualized with a chemiluminescence detection system (Cat#: WBLUR0500) purchased from Millipore.
Wblur0500
Manufactured by Merck Group
Sourced in United States, Germany
WBLUR0500 is a laboratory equipment product. It serves a core function in laboratory operations, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Iseq00010, by Merck Group (2 mentions)
Anti gst antibody, by GenScript (2 mentions)
Anti myc antibody, by GenScript (2 mentions)
Anti flag antibody, by GenScript (2 mentions)
Abcb1 antibody sc 8313, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer high, by Solarbio (1 mentions)
α synuclein, by Cell Signaling Technology (1 mentions)
Multi gauge v3.0 program, by Fujifilm (1 mentions)
Fugene hd, by Promega (1 mentions)
F1804, by Merck Group (1 mentions)
Ab4074, by Abcam (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab6900, by Abcam (1 mentions)
Af5208, by R&D Systems (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Non fat milk, by Bio-Rad (1 mentions)
Ipvh00005, by Merck Group (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
12 protocols using wblur0500
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of Protein Expression
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Cells and lung tissue lysates were extracted with RIPA buffer (high) (Solarbio, R0010, China) containing 1% protease inhibitors (MCE, USA) and a phosphatase inhibitor cocktail. The samples were mixed with SDS lysis buffer (10% SDS, 0.25 M Tris-HCl at pH 6.8, 50% glycerol, 1% β-mercaptoethanol, and bromophenol blue) and separated by SDS‒PAGE. For nonreducing gel analysis, the cells were lysed in 20 mM Tris (pH 7.0) containing 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate, and 1 μg/mL leupeptin and separated by native PAGE. Then, the proteins were transferred to PVDF membranes (Millipore, USA). After being blocked with 5% skim milk or BSA for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight and then with the HRP-labeled secondary antibodies for 1 h at room temperature. The antibodies used for protein expression analysis are listed in Supplementary Table 2 . Bands were visualized with an enhanced chemiluminescence kit (WBLUR0500, Millipore, USA).
3
Comparative Analysis of Parkinson's Protein Markers
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Fifteen μL of the HSW from each sample was loaded on four gradient SDS-protein gels (Bio-Rad, 4–15%; Hercules, CA, USA) so that a gender- and age-matched patient/control pair was loaded side by side. In total, we analyzed 27 PD samples and 27 gender- and age-matched non-PD samples (14 male and 13 female pairs). The neuroblastoma SH-SY5Y cells were harvested after differentiation with 10 μM retinoic acid for 6 days and lysed with lysis buffer (1% triton X-100 in PBS, 1x protease inhibitor cocktail (Genedepot, Barker, TX, USA)). The SH-SY5Y cell lysates were used as a positive control for the western blot analysis. All samples in the gels were processed for Western blot analysis under the same conditions for each antibody: LRRK2 (MJFF2 Abcam, Cambridge, MA, USA; ab133474, 1 : 1000), α-synuclein (Cell Signalling Technology (CST), Danvers, MA, USA; #2642, 1 : 800), DJ-1 (CST, #2134, 1 : 1000), and TSG101 (Abcam, ab83, 1 : 500). Specific bands of the four separate blots were detected with an enhanced chemiluminescence reagent (WBLUR0500, Millipore, Milford, MA, USA) using the Microchemi 4.2 device (DNR Bioimaging Systems, Jerusalem, Israel) under the same conditions and their densities were analyzed with the Multi-Gauge V 3.0 program (Fuji photo Film, Tokyo, Japan).
4
PNPLA2 and PNPLA3 Overexpression in HEK293 Cells
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HEK293 cells (ATCC CRL‐1573) were grown under standard conditions and transfected with FLAG (DDK)‐tagged plasmids expressing human or mouse PNPLA2 or PNPLA3, or VHH‐FLAG‐Halo (Table S1 ). Cells were transfected using Fugene HD (Promega) according to the instructions from the vendor. After 48 hours, cells were lysed and western blot experiments performed as detailed in the Supporting Materials and Methods . The following primary antibodies were used: sheep polyclonal anti‐human PNPLA3 (1:1000, AF5208; R&D Systems, Minneapolis, MN, USA), mouse monoclonal anti‐FLAG M2 (1:1000, F1804; Sigma Aldrich, USA), and rabbit polyclonal anti‐alpha tubulin (1:200, ab4074; Abcam, UK). Membranes were washed and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies anti‐rabbit (1:3000, 7074S; Cell Signaling Technologies, Inc, USA), anti‐mouse (1:3000, 7076S; Cell Signaling Technologies), or anti‐sheep (1:2000, ab6900; Abcam). Western blots were visualized with chemiluminescent HRP substrate (WBLUR0500; Millipore, USA), using the ChemiDoc MP imaging system (Bio‐Rad, USA).
5
Western Blot Protein Detection Protocol
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Western blot was performed, as previously described69 (link). Briefly, cells were washed with cold DPBS twice before lysed with M-PER buffer (Thermo Scientific, #78501) containing proteinase inhibitors cOmplete (Roche, #11836170001) and phosphatase inhibitors (Sigma, #P2850 and P5726). Lysates were then collected and transferred to 1.5 mL Eppendorf tubes and sonicated. Protein concentration was determined by NanoDrop. Fifty μg of total proteins were loaded onto each lane of an 8–12% SDS-PAGE gel. After electrophoresis, proteins on the gel were transferred to 0.45 μm of PVDF membrane (Millipore, #IPVH00005) in a sponge sandwich. Membranes were then blocked with 5% of non-fat milk (Bio-Rad, #170–6404) and probed with primary antibodies overnight on a shaker in cold room. Membranes were then washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were then incubated with Western HRP substrate (Millipore, WBLUR0500) for 2–5 min before imaging with an X-ray film. The antibodies used for WB are listed in Supplemental Table. β-actin was blot as loading control on the same gel with proteins of interest. Uncropped and unprocessed scans of all blots were provided in the Source Data file.
6
Quantitative Western Blot Analysis of Caspase 3
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BXPC-3 cells were incubated at 37 °C under 5% CO2 atmosphere. For quantitative Western blot analysis, 70–80% confluent cells were seeded at 2 × 105/per well onto 6-well plates for 12 h, followed by stimulation with 2.5 μM, 3.0 μM 1 for 24 h. The cultured cells were first washed twice with precooled PBS, followed by the addition of a RIPA lysis buffer combined with a mixture of proteases or phosphatase inhibitors to lyse the total protein, and then the protein concentration was quantified by a BCA protein assay kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Equal amounts of protein extract were separated on a 12% SDS-PAGE gel and electrotransferred to 0.22 mm PVDF membranes using a Bio-Rad wet transfer tank. After blocking with 5% nonfat milk at room temperature for 2 h, membranes were incubated with the specific antibodies targeting caspase 3 (wanlei, 1:1000) and β-actin (Affinity, 1:1000) at 4 °C overnight. After incubating with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (CST), protein bands were detected using an enhanced chemiluminescence kit (Millipore, WBLUR0500) and imaged. Band intensities were quantified using Image J software.
7
Western Blot Analysis of Protein
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Total protein of DRG and spinal cord were prepared and subjected to western blot as mentioned in our previous study (Zhang et al., 2014) . Membranes were incubated overnight at 4 C with primary antibody followed by incubation with HRP-conjugated secondary antibody for 2 hours at room temperature. Signals were developed using the western HRP substrate (WBLUR0500, Millipore).
8
Western Blot Analysis of Cellular Proteins
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Cell lysates and exosome pellets were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). 40 μg of total protein were denatured in electrophoresis sample buffer and boiled at 95°C for 5 min before running SDS-PAGE. Afterward, proteins were transferred into polyvinylidene fluoride (PVDF) membranes. After blocking, the PVDF membranes were probed with antibodies for actin (1:5,000, Thermo Fisher, MA5-11869, USA), cytochrome c (1:5,000, Proteintech, 10993-1-AP, USA), HSP70 (1:5000, Bio-rad, MCA6096, USA) at 4°C for 12 h. After several thorough washes with Tween-Trisbuffered saline (TTBS), the PVDF membranes were incubated with the secondary antibodies in TTBS at room temperature for 1 h. After washing with TTBS, the PVDF membranes were visualized using Immobilon Crescendo Western horseradish peroxidase (HRP) substrate (Merck, WBLUR0500), according to the manufacturer's instructions.
9
Exploring NF-κB Signaling in Breast Cancer
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MDA-MB-231 cells that were continuously treated by TNFα + IL-1β/vehicle were cultured overnight in complete media supplemented with TNFα + IL-1β/vehicle. In specific experiments (as indicated), the media were replaced with fresh DMEM containing 0.5% FBS and were supplemented with OXPHOS inhibitors (Rot+AA) or glycolysis inhibitor (2-DG) (concentrations as in ELISA assays), without TNFα + IL-1β/vehicle. Control cells were supplemented with the inhibitor solubilizer. After 2 h, TNFα + IL-1β/vehicle were added to the cells without a wash, and lysates of cells were made after 15 min or 24 h using RIPA buffer. Total-p65 and phosphorylated p65 (p-p65) were detected using specific antibodies (#8242 and #3033, respectively; Cell Signaling Technology, Danvers, MA, USA). GAPDH was used as loading control (detected by antibodies #ab9485, Abcam). The membranes were reacted with streptavidin-horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#111-035-003, Jackson Immunoresearch laboratories), and subjected to ECL analysis (#WBLUR0500, Merck, Darmstadt, Germany).
10
Western Blot Analysis of STAT and NF-κB Signaling
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MCF-7 cells have been TME-stimulated or treated by vehicles (as described above) for different time points (as described in Figure legends). Following lysis in RIPA buffer, Western blot (WB) was performed using antibodies from Cell Signaling Technology (CST, Danvers, MA, USA), except where otherwise indicated: Total (T)-STAT3: (#4904); Phosphorylated (P)-STAT3 Y705: (#9145); (P)-STAT3 S727: #9134; (T)-STAT1: #9172; (P)-STAT1: #9167; (T)-p65: #8242; (P)-p65: #3033. GAPDH (#ab9485; Abcam, Cambridge, UK) or β-Tubulin (#2146) served as loading controls. Then, the membranes were incubated with streptavidin-horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#111-035-003; Jackson ImmunoResearch Laboratories). The membranes were subjected to enhanced chemiluminescence (ECL) analysis (#WBLUR0500, Merck, Darmstadt, Germany), and were visualized with Amersham ImageQuant 800 (GE Healthcare, Little Chalfont, UK). Quantification was carried out using ImageJ software (version 1.4).
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