To obtain cell lysates, cells were washed with ice‐cold PBS and incubated and harvested with a lysis buffer (150 mmol/L NaCl, 10 mmol/L NaH2PO4, 1 mmol/L EDTA, 1% TritonX100, 0.5% SDS) with protease inhibitor cocktail and phosphatase inhibitor (Sigma). Mouse and human brain were homogenized in RIPA buffer (ThermoFisher) with protease inhibitor cocktail and phosphatase inhibitor (Sigma) and used for Western blotting as described.9 The following primary antibodies (dilutions) were used: anti‐KCa3.1 P4997 (1:1000, Sigma), anti‐phospho P38MAPK and anti‐P38MAPK (1:1000, Cell Signaling), anti‐β actin (1:3000, Sigma), and anti‐GAPDH (1:2000, Cell Signaling). Secondary antibodies were HRP‐conjugated anti‐rabbit or anti‐mouse antibody (1:1000, GE Healthcare). Brain tissue samples from 5xFAD treated with senicapoc or control diet were fractionated into TBS‐soluble and TBS‐insoluble, SDS‐soluble fractions, which were used for Aβ42 quantification by a Human Aβ42 ELISA kit (Wako) as described.16 Alternatively, Aβ in each fraction was separated in 16.5% Tris/Tricine SDS gel electrophoresis (Bio‐Rad), transferred to PVDF membrane, and detected by anti‐Aβ (6E10, 1:1000, BioLegend).
Protease inhibitor and phosphatase inhibitor cocktail
Manufactured by Merck Group
Sourced in United States, China, Germany, Canada
Protease inhibitor and phosphatase inhibitor cocktails are laboratory reagents used to prevent the degradation of proteins and protein modifications during sample preparation and analysis. These cocktails contain a mixture of chemical compounds that inhibit the activity of various proteases and phosphatases, which can help preserve the integrity of proteins and protein phosphorylation states in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (9 mentions)
Ripa buffer, by Merck Group (8 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Ripa buffer, by Cell Signaling Technology (5 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Protein extraction kit, by Keygen Biotech (3 mentions)
β actin, by Merck Group (3 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Hrp conjugated anti rabbit or anti mouse antibody, by GE Healthcare (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Foxo1, by Cell Signaling Technology (2 mentions)
Ecl solution, by Cytiva (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
89 protocols using protease inhibitor and phosphatase inhibitor cocktail
1
Cell Lysis and Western Blotting for Alzheimer's
2
DNA-Protein Interaction Analysis
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Cell pellets from MM or MM-3D cells were lysed with CellLytic M (Sigma) with protease inhibitor and phosphatase inhibitor cocktails (Millipore, Etobicoke, ON, Canada) on ice for 30 min. 300 µg of total cell lysate was mixed with 3 pmol of STAT3 DNA probe (Biotin-5′-GATCTAGGAATTCCCAGAAGG-3′) for 30 min on a rotator at room temperature. 75 µL of streptavadin agarose beads (Thermo Scientific, Ottawa, ON, Canada) was added to the DNA-lysate mix. The whole solution was incubated on a rotator at 4 °C overnight. The beads were washed three times with ice-cold PBS. SDS loading buffer was added to beads and boiled for 5 min to dissociate bound proteins. The beads were spun down and the supernatant was subject to SDS-PAGE.
3
Western Blot Analysis of Signaling Pathways
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Both MM and MM-3D cell pellets were lysed by 1× RIPA buffer (Millipore) with protease inhibitor and phosphatase inhibitor cocktails (Millipore) on ice for 30 min. Protein concentration of each lysate was measured using BCA protein assay kit (Thermo Scientific). Equal amount of protein was loaded on 10% homemade polyacrylamide gels for SDS-PAGE at 100 volts. Proteins in polyacrylamide gel were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) at 100 V for 2 h. Primary antibodies used were anti-pSTAT3 (Y705) (1:2000, CST, #9145), anti-STAT3 (1:1000, CST, #124H6), anti-pErk (T202/Y204) (1:2000, CST, #4377), anti-Erk (1:1000, Enzo, #ADI-KAP-MA001), anti-pAkt (S473) (1:1000, CST, #4060), anti-Akt (1:1000, CST, #9272), anti-cleaved (V1744) Notch1 (1:1000, CST, #4147), anti-Notch1 (1:1000, CST, #3439), anti-pIKBα (S32) (1:1000, CST, #9241), anti-IKBα (1:1000, CST, #4812), anti-β-actin (1:1000, CST, #58169), anti-PARP (1:1000, CST, #9532) and anti-Caspase3 (1:1000, CST, #9665). Secondary antibodies used were HRP-conjugated anti-mouse (1:2000, CST, #7076) and anti-rabbit (1:2000, CST, #7074). Signals on the membrane were developed using Pierce™ ECL Western Blotting Substrate (Thermo Scientific) and exposed to X-ray films (Fuji, Tokyo, Japan).
4
Liver and Spleen Tissue Lysis and Western Blotting
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Liver and spleen lysates were prepared by dounce homogenization of 80 mg fresh liver or spleen tissue in 1 mL radioimmunoprecipitation assay (RIPA) buffer with added protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich) as previously described [24 (link)]. For testing the specificity of GalNac knockdown, GalNAc-Control-ASO and GalNac-C3-ASO-treated livers underwent collagenase perfusion to isolate the non-parenchymal cell (NPC) and hepatocyte compartments prior to homogenization as previously described [24 (link)]. Supernatant protein concentration was measured using a Bradford protein spectrophotometric assay kit (Bio-Rad) followed by Western Blotting as previously described [24 (link)]. The membranes were then incubated with the desired primary antibodies overnight followed by secondary antibody (Cell Signaling Technology) for 1 h. Finally, the proteins were visualized using Luminol enhanced chemiluminescent (ECL) reagent (Cat# 34095, Thermo Fisher Scientific) on a ChemiDoc MP Imaging System (Bio-Rad). Image analysis, densitometry, and gray level calculation for each band in pixels normalized to a reference band (depicted under WB images) was performed using Image Lab Software with background subtracted per the Bio-Rad manual. Full-length original western blots are available in the supplemental materials section.
5
Analyzing β-catenin Levels after Retinal Surgery
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Ten days after surgery (n = 3 eyes/group), retinas were harvested and lysed in RIPA buffer, with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich, Gillingham, UK). Eluted proteins samples (20 μg) were run on 10% (wt/vol.) SDS-PAGE gels. Samples were probed using rabbit anti-β-catenin (1:2000; Cell Signaling Technology, Danvers, MA, USA) and protein levels compared with β-actin (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA). Antibodies were validated by the manufacturers for western blot analysis (ESM Table 2 ).
6
SDS-PAGE and Western Blotting Analysis
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SDS‐PAGE and subsequent western blotting analyses were performed as previously described.29 Cell lysates were prepared by solubilizing cells in radio‐immunoprecipitation assay (RIPA) buffer (50 mM Tris‐HCl [pH 7.5] containing 150 mM NaCl, 1% NP‐40, and 0.1% sodium deoxycholate) supplemented with protease‐inhibitor and phosphatase‐inhibitor cocktails (Sigma). Samples were then mixed at a 1:4 ration with 5 × sample buffer (300 mM Tris‐HCl [pH 6.8] containing 10% 2‐mercaptoethanol, 10% SDS, 50% glycerol and 0.05% Coomassie Brilliant Blue) and boiled for 5 min. The resulting samples (20 μg protein/lane) were subsequently separated by SDS‐PAGE using pre‐cast 5%–20% Tris‐glycine gradient gels (Atto Corporation, Tokyo, Japan) and transferred to PVDF membranes (Wako Pure Chemical). Membranes were blocked by incubation with TBS containing 0.1% Tween 20 (TBS‐T) and 5% skim milk (Megmilk Snow Bland, Tokyo, Japan) or BSA (Sigma) for 1 h, probed with the appropriate primary antibodies diluted with Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) at 4°C, washed extensively with TBS‐T, and incubated with HRP‐conjugated secondary antibodies (1:5000 dilution) (GE Healthcare, Little Chalfont, UK). Finally, the membranes were washed, developed by incubation with ECL detection reagents (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare).
7
Immunoblot Analysis of Cell Lysates
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Immunoblots were performed as previously described [17 (link)]. Briefly, cells were lysed in lysis buffer (35 mM Tris [pH 7.4], 0.4 mM EGTA, 10 mM MgCl2, and 0.1% Triton-100) containing protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). The total cell lysate was homogenized in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled, subjected to SDS-polyacrylamide (10%) gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 1% BSA and incubated with the primary antibodies. After it was rinsed with 0.1% Tween-20 in PBS, the membrane was incubated with the appropriate HRP-conjugated secondary antibody. The intensity of the positive signals was visualized by chemiluminescence (GE Healthcare, Buckinghamshire, UK), and the images were imported by Image Reader LAS-1000 Plus (Fuji Photo Film Co. Ltd., Tokyo, Japan).
8
Maintaining and Transfecting Cell Lines
9
Protein Expression Analysis in Kidney Samples
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Protein samples from kidney tissue or cell culture were lysed in radioimmunoprecipitation assay buffer with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysates were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% BSA/TBS-0.5% Tween-20 followed by incubation with primary antibodies overnight at 4°C. For immunoprecipitation, tissue lysates were incubated with anti-TIE2 antibody and subsequently with Dynabeads against Protein A (Invitrogen). The samples were eluted with Laemmli sample buffer with a reducing agent containing 200 mM dithiothreitol. The following antibodies were used for Western blot analysis: TIE2 antibody (Santa Cruz; C-20; SC-324), 4G-10 platinum anti-phosphotyrosine (EMD Millipore; 05–1050), α-tubulin antibody (Genscript; A01410), eNOS (Cell Signaling Technology; 49G3), phospho-eNOS (Cell Signaling Technology; C9C3), and FOXO1 (Cell Signaling Technology; C29H4). HRP-tagged secondary antibodies (Jackson ImmunoResearch) were used in a dilution of 1:10,000 in TBS-0.5% Tween. Quantification of protein expression was performed on multiple replicate experiments using the software ImageJ.
10
Protein Extraction and Western Blot Analysis
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Western blot was performed according to the method described previously [30 (link)]. Briefly, cytoplasmic and nuclear proteins were extracted from the kidney tissue using the Protein Extraction Kit (KeyGen Biotech Co. Ltd., Nanjing, China), containing protease inhibitor and phosphatase inhibitor cocktails (Sigma Aldrich, Darmstadt, Germany), according to the manufacturer’s protocols. The total protein extracted concentration was considered using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Afterward, 50 µg of the total extracted protein was separated via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in Tris-buffered saline (TBS), containing 3% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature. After washing with TBS containing 0.1% Tween 20, the membranes were incubated firstly with the primary antibodies (1:300 dilution) for 2 h, and then goat antirabbit HRP-conjugated (as secondary antibody; at a 1:5.000 dilution) at room temperature. The chemiluminescence produced from the luminol reagent was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.
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