The total proteins in cell supernatants were concentrated using Amicon Ultra-15 Centrifugal Filters (Millipore Canada, Etobicoke, ON, Canada) according to the manufacturer’s instructions. The concentrations of protein in cell lysates and supernatants were determined using Quick Start Bradford Protein Assay. Equal protein amounts were separated on 10% SDS-PAGE gels and then transferred to nitrocellulose membranes (GE HealthCare Life Sciences, Mississauga, ON, Canada) and blocked using 5% BSA (Sigma Aldrich). Primary antibodies were used at a dilution of 1:1,000 goat anti-caspase-1 (R&D Systems); rabbit anti-cleaved caspase-1 (p20) or mouse anti-α-tubulin (Cell Signaling Technology, Boston, MA, USA). Secondary antibodies were used at a dilution of 1:1,000 HRP-conjugated rabbit anti-goat (R&D systems) or 1:5,000 HRP-conjugated rabbit anti-mouse or mouse anti-rabbit (Cell Signaling Technology). Signal detection was carried out using the West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific).
West pico plus chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The West Pico PLUS Chemiluminescent Substrate is a laboratory reagent used for the detection and quantification of proteins in western blot analysis. It produces a chemiluminescent signal that can be captured and measured by a suitable detection system.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Wuhan Servicebio Technology (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
0.45 μm pvdf membrane, by Merck Group (2 mentions)
Hrp linked anti rabbit secondary antibody, by Cell Signaling Technology (2 mentions)
Pstat1 tyr701, by Cell Signaling Technology (2 mentions)
P stat3 tyr705, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Nupage, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Biomax film, by Kodak (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
27 protocols using west pico plus chemiluminescent substrate
1
Protein Concentration and Western Blot Analysis
2
FOXA1 Expression in H2.35 Cells
3
SiMPull Assay and Western Blotting
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For SiMPull assays, the cells were collected after 24-hr transfection in detergent-free buffer (40 mM HEPES, pH8.0, 150 mM NaCl, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 2 mM EDTA, 1x Sigma protease inhibitor cocktail). The cells were lysed by sonication for 3 seconds on ice followed by ultracentrifugation at 90,000 × g in a TLA100.3 rotor for 1 h at 4 °C. For SiMPull, EGFP concentration was measured using a standard emission (ex488/em520) curve of pure recombinant EGFP (see Supplementary Fig. 1B ), and each cell lysate was diluted in vesicle buffer (10 mM Tris·HCl, pH 8.0, 150 mM NaCl) to yield 5 nM EGFP. For western blotting, cells were lysed in 1x SDS sample buffer or as described above and mixed at 1:1 with 2x SDS sample buffer, both containing β-mercaptoethanol at a final concentration of 5%, and heated for 5 min at 95 °C. Proteins were resolved by SDS-PAGE and transferred onto PVDF membrane. The membrane was incubated with primary and secondary antibodies following manufacturers’ recommendations. HRP-conjugated secondary antibody was reacted with West Pico PLUS Chemiluminescent Substrate, and the signal was detected using an iBright CL 1000 imaging system (Thermo Fisher Scientific).
4
Western Blot Analysis of TNBC Cells
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Initially, the TNBC cells were lysed and the proteins were extracted using RIPA buffer (P0013B, Beyotime). Then, the proteins were separated through 10–15% SDS-PAGE and blotted onto 0.45 μm PVDF membranes (Millipore). Subsequently, the membranes were then underwent overnight incubation at 4 °C with primary antibodies as follows: anti-β-actin antibody (1:2000, 20536-1-AP, Proteintech), anti-SLC25A17 antibody (1:1000, A14840, Abclonal), anti-STAT3 (1:1000, #9139, CST), anti- Phospho-STAT3 (1:1000, #9145, CST), anti-JAK2 (1:1000, #3230, CST), anti-Phospho-JAK2 (1:1000, #3771, CST), anti-Ecadherin (1:20000, 20874-1-AP, Proteintech), anti-Vimentin (1:20000, 60330-1-Ig, Proteintech), anti-MMP2 (1:1000, 10373-2-AP, Proteintech), anti-MMP9 (1:1000, 10375-2-AP, Proteintech), anti- Cleaved PARP (1:1000, #5625, CST), anti-Cleaved Caspase-3 (1:1000, #9664, CST), anti-LC3 (1:1000, T55992, Abmart), anti-P62 (1:5000, T55546, Abmart), anti-Beclin-1 (1:1000, T55092, Abmart), anti-Bcl-2 (1:1000, T40056, Abmart), anti-Bax (1:1000, T40051, Abmart). Following primary antibody incubation, the membranes were exposed to secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized using the West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific).
5
Quantitative Western Blot Analysis
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After induction with IL-10 (30 ng/ml) for the indicated time points, 2 × 105 cells were lysed in 80 µl of 2x SDS sample buffer (Bio-Rad), separated on a precast NuPAGE 4–12% gradient gel (Life Technologies) and transferred onto a nitrocellulose membrane (Bio-Rad). Blocking was performed for 1 h in TBS containing 0.1% Tween 20 and 5% nonfat dry milk. Antibodies against p-STAT1-Tyr701 (Cat no. 9167), STAT1 (Cat no. 9172), p-STAT3-Tyr705 (Cat no. 9145), STAT3 (Cat no. 4904), β-Actin (Cat no. 4970), SOCS1 (Cat nos 3957 and 3950), SOCS2 (Cat no. 2779), SSH1 (Cat no. 13578) and HRP-linked anti-rabbit secondary antibody (Cat no. 7074) were all purchased from Cell Signaling Technology and used according to the manufacturer’s instructions. West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and BioMax film (Kodak) were used for detection. ImageJ (NIH) was used for quantification of Western Blots.
6
Western Blot Analysis of IGF2BP3 Protein
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Total cellular protein was extracted with RIPA lysis buffer (Servicebio, Wuhan, China) containing phosphorylase and protease inhibitors (Servicebio, Wuhan, China) and then denatured by mixing 5× loading buffer (Servicebio, Wuhan, China) and boiling for 10 min. Then the denatured protein was separated by electrophoresis in 10% SDS-PAGE gels and transferred to PVDF membranes (Sigma, Darmstadt, Germany). The membranes were blocked in 5% BSA (Servicebio, Wuhan, China) for 1 h at room temperature and then incubated with the following primary antibodies: IGF2BP3 (Abclonal, Wuhan, China, CAT# A6099, 1:1000) and GAPDH (Huabio, CAT# ET1601-4, 1:5000) at 4 °C overnight. Then the membranes were washed with TBST 10 min for three times and incubated with the secondary antibody (Promotor, Wuhan, China, CAT# HA1005, 1:5000) at room temperature for 1 h. Finally, the proteins were visualized with West Pico plus Chemiluminescent Substrate (Thermo Fisher Scientific, Carlsbad, CA, USA).
7
SYK-LILRB2 Signaling Pathway Analysis
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HMC3 cells were seeded into a 6-well plate at 1 × 106 cells/well in EMEM without FBS. After 1-hour incubation with designated treatments, the supernatant was removed, and cells were washed three times by DPBS. The cell lysate was obtained by lysing cells using NP-40 lysis buffer (1% NP40, 50 mM Tris-HCl, pH = 8, 150 mM NaCl) with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (ThermoFisher). After removing debris by centrifugation, the total protein amount normalized by Pierce BCA Protein Assay Kit (ThermoFisher). Protein samples were resolved by 10% SDS-polyacrylamide gels (Biorad) and later transferred onto Immun-Blot PVDF membranes (Biorad). Proteins were probed with specific primary antibodies and secondary antibodies diluted in 5% BSA TBST [21 (link), 24 (link), 29 (link)]. The primary antibodies used are: SYK (1:1000, Cell Signaling 13198S), Phospho-Syk (Tyr525/526) (1:1000, Thermo Fisher MA5-14918), β-Actin (1:1000, Cell Signaling 4970S), SHP1 (1:1000, Cell Signaling 3759S), Phospho-SHP-1 (Tyr564) (1:1000, Cell Signaling 8849S), and LILRB2 (1:1000, Thermo Fisher PA5-46983).
The immunoreactive bands were visualized with the West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The immunoreactive bands were quantified using ImageJ. Three independent treatment replicates were conducted with the representative immunoblot shown.
The immunoreactive bands were visualized with the West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The immunoreactive bands were quantified using ImageJ. Three independent treatment replicates were conducted with the representative immunoblot shown.
8
Western Blot Analysis of PRC2 Complex
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Actively growing cultures were collected and lysed with RIPA buffer (0.5% deoxycholate, 1% IGEPAL-CA630, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris-8.1), cleared by centrifugation, and quantified for protein content using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Hundred micrograms of protein extracts were prepared for SDS-PAGE by boiling with reducing agents, loaded in equal proportions for separation on 4 to 15% acrylamide gels (Bio-Rad, #456-1086), and transferred to nitrocellulose membranes (Cytiva, #10600002). Antibodies used for western blots were as follows: EZH2 (Cell Signaling, #5246s, 1:1000), H3K27me3 (Cell Signaling, #9733s, 1:1000), SUZ12 (Active Motif, #39357, 1:1000), EED (Millipore, #09-774, 1:1000). They were incubated with the membranes overnight at 4 °C. Histone H3 (AbCAM, Cat# ab1791, 1:5000) was used as loading control. After washing, membranes were incubated in secondary anti-rabbit IgG, HRP-linked antibody (Novus #NB7160, 1:10,000) for 1 h at room temperature. After washing, chemiluminescence was visualized with West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, #34077) and exposure onto Amersham Hyperfilm ECL (Cytiva, #28-9068-36).
9
Western Blot Analysis of SOCS2 in Stimulated DCs
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Cell pellets of stimulated DCs were harvested and lysed in 60 µL of 2× Laemmli sample buffer (BIO-RAD, Leipzig, Germany) supplemented with 5% β-mercaptoethanol (Sigma-Aldrich). Subsequently, probes were separated on a 4%–12% gradient gel (NuPAGE, Life Technologies, Vienna, Austria) and blotted onto a nitrocellulose membrane (BIO-RAD), which was then blocked in TBS supplemented with 0.1% Tween and 5% nonfat dry milk for 1 h. The following antibodies were used according to the manufacturer’s instructions: SOCS2 (Cat no. 2779, Cell Signaling, Leiden, Netherlands) and HRP-linked anti-rabbit secondary antibody (Cat no. 7074S, Cell Signaling). Detection was done using West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and BioMax films (Kodak, Sigma-Aldrich, Vienna, Austria). Blots were quantified using ImageJ (NIH) software [33 (link)].
10
Chk1 and PARP Cleavage Assay
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Protein expression of Chk1 (Cell signaling technology #2360) and p-Chk1(S345) (Cell signaling technology, #2348) was determined in JJ012, SW1353 and CH2879 in control conditions and after treatment for 2 or 24 h with IC50 concentrations of LY2603618 (JJ012 1 µM, SW1353 441 nM, CH2879 449 nM). In addition PARP cleavage (Cell signaling technology #9532) was assessed after treatment of JJ012, SW1353 and CH2879 for 2 or 24 h with IC50 concentrations of MK-5108, LY2603618 or Volastertib (MK-5108; SW1353: 1 µM, JJ012: 513 nM, CH2879: 847 nM, Volasertib: SW1353: 34 nM, JJ012 11 nM, CH2879: 24 nM, LY2603618 as described above). Lysates were obtained of cells grown until 70% confluence using hot-SDS buffer (1% SDS, 10 mM Tris/EDTA with complete inhibitor (Roche #11697498001) and phosSTOP (Roche #04906837001) as previously described [4] (link). Expression of gapdh (Cell signaling technology #5174) was determined as a loading control. A total of 10 µg was loaded on the gel for each sample and blocking was performed using 5% milk. Primary antibodies were diluted in 5% BSA (bovine serum albumin) and incubated overnight. Blotting was performed on PVDF membranes and detection was done using enhanced chemo-luminescence (west Pico Plus chemiluminescent Substrate, Thermo Fisher Scientific, Waltham, MA, USA) followed by visualization using the ChemiDoc imaging system of Biorad.
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