The markers (CD63 and Alix) of the isolated sEVs were correspondingly detected by Western blotting as reported previously.62 , 63 The sEVs were lysed using the Mammalian Cell Lysis kit (Sigma–Aldrich) and quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). The samples were preheated at 60°C for 15 min. Proteins were electrophoresed in an SDS‐polyacrylamide gel followed by transferring to PVDF membranes. The membranes were blocked with 5% BSA in TBS‐Tween 20 and were then probed with antibodies specific for CD63 (1:1000, ab216130, Abcam), Alix antibody (1:1000, Proteintech), and calnexin (1:1000, Proteintech, negative biomarker for sEVs) overnight at 4°C. After three washes in TBS‐tween 20, membranes were incubated with the secondary antibody (Thermo Scientific) for 1 h and washed again. For visualization, blots were exposed to SuperSignal West Dura Extended Duration substrate and measured by the FluorChem R system (ProteinSimple).
Mammalian cell lysis kit
Manufactured by Merck Group
Sourced in United States, Macao
The Mammalian Cell Lysis kit is a laboratory tool designed to facilitate the extraction of cellular contents from mammalian cells. It provides a standardized and efficient method for the disruption of cell membranes, allowing for the recovery of intracellular components such as proteins, nucleic acids, and other biomolecules. The kit includes reagents and protocols to ensure consistent and reliable results.
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Lab products found in correlation
β actin, by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Fla 5100, by Fujifilm (6 mentions)
Ab216130, by Abcam (5 mentions)
A5316, by Merck Group (4 mentions)
Ipvh00010, by Merck Group (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Supersignal west dura extended duration, by Thermo Fisher Scientific (3 mentions)
Pico plus chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Ab16502, by Abcam (3 mentions)
Goat anti mouse rabbit igg, by Jackson ImmunoResearch (2 mentions)
Enhanced chemiluminescent reagent, by Cytiva (2 mentions)
Nupage mops sds buffer kit, by Thermo Fisher Scientific (2 mentions)
Anti gapdh antibody, by Abcam (2 mentions)
Protein a g bead, by Santa Cruz Biotechnology (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Glial fibrillary acidic protein (gfap), by Santa Cruz Biotechnology (2 mentions)
51 protocols using mammalian cell lysis kit
1
Western Blot Analysis of sEV Markers
2
Mammalian Cell Lysis and Western Blot Analysis
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Treated cells or isolated MIVs were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich), as described previously (Niu et al., 2014 (link)). Equal amounts of protein were electrophoresed in a SDS-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes. Blots were blocked with 5% BSA in TBS-Tween, and Western blots were probed with antibodies specific for σ-1R (15168-I-AP; Proteintech Group), Src (2108; Cell Signaling), p-PDGFR-β (3161; Cell Signaling), NG2 antibodies (ab129051; Abcam), TBX18 (ab115262; Abcam), αSM (A5228; Sigma-Aldrich), and histone H3 (9715; Cell Signaling) at 1:1,000 dilution; NF-κB (ab16502; Abcam) at 1:2,000 dilution; ganglioside GM1 (bs-2367R; One World Lab) at 1:500 dilution; and p-Src (ab32078; Abcam), PDGFR-β (ab32570; Abcam), Desmin (ab32362; Abcam), and β-actin (A5316; Sigma-Aldrich) at 1:5,000 dilution. Secondary antibodies were alkaline phosphatase conjugated to goat anti-mouse/rabbit IgG, or rabbit anti-goat IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). All experiments had at least four biological replicates, and representative blots are presented in the figures.
3
Western Blot Analysis of Protein Samples
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Protein was isolated from harvested cells using mechanical disruption and the Mammalian Cell Lysis Kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s instruction. The proteins were separated on 1 mm NuPage Novex 10% Bis-Tris gels using the NuPage MOPS SDS Buffer Kit (Life Technologies, Carlsbad, CA, USA) followed by electrotransfer to 0.2 mm nitrocellulose membranes (Pall, Port Washington, WI, USA). Nonspecific binding sites were blocked with 5% bovine serum albumin in PBS for 1 hour at room temperature (RT). Membranes were then incubated with diluted primary antibody (1:1000; Cell Signaling Technology, Inc.) at 4°C overnight. Following three washes with PBS containing 0.5% Tween-20, membranes were incubated with diluted secondary antibody (GE Healthcare, Buckinghamshire, UK) at RT for two hours. The signal was visualized with an enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ). For the protein loading control, blots were stripped and stained for GAPDH using an anti-GAPDH antibody (1:2000, AbCam, Cambridge, MA).
4
Immunoprecipitation of TLR2 in Cocaine-Treated BV2 Cells
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Immunoprecipitation was performed as described previously [21 , 22 (link)]. BV2 cells were treated with cocaine (10 μM) for 1 h and then lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich). For each sample, 600 μg of protein was used for immunoprecipitation. Cell lysates were incubated with TLR2 antibody overnight at 4 °C followed by incubation with 30 μl of protein A/G beads (Santa Cruz, 2003) for 1.5 h at 4 °C. The mixture was then centrifuged at 12,000 rpm for 1 min, and the cell pellets were rinsed twice with the lysis buffer (1.0 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4) containing proteinase and phosphatase inhibitors. Finally, 30 μl of 2 × western blot loading buffer was added and boiled for 5 min. Then, the protein complexes were detected using 4G10 antibody (Millipore, Cat# ab5320). Input protein (without antibody addition) served as a control to demonstrate that equal amount of total protein was used.
5
Western Blot Analysis of Astrocyte Proteins
6
Glioblastoma CSCs Lysis and Protein Extraction
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Glioblastoma CSCs consisting of U87 CD133+ NSCs and HSCs, were lysed using a Mammalian Cell Lysis kit (Sigma-Aldrich; Merck Millipore) according to the manufacturer's instructions. Briefly, cells were collected transferred to a conical test tube and centrifuged at 25°C for 5 min at 420 × g. The supernatant was decanted and the cells were washed twice by resuspending the cell pellets in PBS and centrifuging at 420 × g for 5 min at 25°C. After decanting the supernatant, cells were resuspended in cell lysis buffer (106-107 cells/ml) and incubated for 15 min on an orbital shaker. The cells were subsequently centrifuged at 12,000 × g for 10 min at 25°C to pellet the cellular debris. The protein-containing supernatant was removed and transferred to a fresh test tube. Samples that were used immediately were incubated on ice, while the remaining samples were stored at −20°C.
7
Western Blot Protein Analysis
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Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich). Equal amounts of protein were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes (Millipore, IPVH00010). The blots were blocked with 5 % nonfat dry milk in PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO). The western blots were then probed with respective antibodies. The protein amounts loaded were normalized according to the β-actin signal using Mouse Anti-β-Actin antibody (Sigma-Aldrich). The secondary antibodies were HRP conjugated to goat anti-mouse/rabbit IgG (Santa Cruz, sc-2005 and sc-2004).
8
Western Blot Analysis of Pericyte Signaling
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Pericytes exposed to different treatments were harvested and total protein was extracted using the Mammalian Cell Lysis kit (Sigma, St. Louis, MO). Nuclear lysates were isolated using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL). Equal amounts of the proteins were run on a sodium dodecyl sulfate-polyacrylamide gel (12%) under reducing conditions. The proteins were then transferred to PVDF membranes and were blocked with 5% non-fat dry milk in PBS. The membranes were then incubated with antibodies recognizing VEGF (Santa Cruz Biotechnology, Dallas, TX, 1:200), the phosphorylated forms of ERK, JNK, p38 and Akt (Cell Signaling, Danvers, MA 1:200), NF-κB p65 (Cell Signaling, 1:1000). Following washing three times, the membranes were then incubated with goat anti-mouse/rabbit secondary antibodies conjugated with horseradish peroxidase (1:5000). Signals were detected by chemiluminescence and imaged on the Microchemi 4.2 (DNR, Israel) digital image scanner according to our previous studies [31 (link),32 (link),37 (link)]. Quantification was performed by densitometry using Image J software (NIH).
9
Autophagy regulation by eNOS signaling
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Tunicamycin, chloroquine, N-acetyl cysteine (NAC), DETA-NONOate, mammalian cell lysis kit, Rabbit anti-actin antibody, DMEM:F12 medium, Mission esiRNA for SQSTM1, and eNOS siRNA were purchased from Sigma-Aldrich. Glycan Differentiation kit, WST-1 cell viability kit, and X-tremeGENE HP DNA Transfection reagent were obtained from Roche. ROS detection kit, gold antifade mounting medium, NuPAGE Novex 4-12% Bis-Tris gels, goat anti-rabbit IgG-alexa 488, Goat-anti-rabbit IgG-Alexa Fluor 568, First Strand cDNA synthesis kit, Fetal Bovine Serum were from Invitrogen. JC1 mitochondrial membrane potential assay kit was from Cayman. Apodirect TUNEL assay kit was purchased from Millipore. Ultralow attachment tissue culture plate was from Nunc. TurboFect siRNA Transfection Reagent, DAPI, and HRP chemiluminescence substrate were from Thermo Scientific. Anti-ubiquitin antibody (clone P4D1) was from Biolegend. Anti rabbit antibodies against LC3, p62, and calnexin, Rabbit mAb phosphor-p70 S6 Kinase (Thr 389), mouse anti-eNOS mAb were obtained from Cell signaling Technology. pcDNA3-eNOS-GFP was obtained from Addgene. LC3-GFP and p62-EGFP constructs were kind gifts from Dr. Beth Levine (Columbia University, NY) and Dr. Terje Johansen (University of Tromso, Norway), respectively.
10
siRNA Transfection of SH-SY5Y Cells
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SH‐SY5Y cells grown in 12‐well plates and 8 well chambers until 60% confluency was subjected to small interfering RNA (siRNA) transfection according to manufacturer's instructions. In short, Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) together with 40 nM ONTARGETplus non‐targeting, Human AMY2A SMARTpool siRNA (Horizon Discovery, Cambridge, UK) were diluted in OptiMEM (Thermo Fisher Scientific, Waltham, MA) and added to the cells. After 24 h, 50% of the medium containing Lipofectamine and siRNA:s was discarded and refilled with the same volume of freshly prepared siRNA and lipofectamine and incubated for additional 24 h. After incubation, the medium was collected and the transfected SH‐SY5Y cells in 12‐well plates were lysed with mammalian cell lysis kit (Sigma‐Aldrich, St. Louis, MO, USA) containing phosphatase inhibitor cocktail 2 and 3 (Sigma‐Aldrich, St. Louis, MO, USA). Cells in 8‐well glass chambers were fixed in 2% PFA. The experiment was repeated three times with two replicates for each condition.
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