Neuro-2a cells were plated onto poly-D-lysine (Sigma-Aldrich) coated six-well plates and transfected as described above and harvested for Western blotting at 72 h after siRNA transfection. Neuro-2a cells were lysed in CytoBuster (Millipore) protease extraction reagent containing protease inhibitors (Roche) as per the manufacturer’s protocol. Cell lysates were separated by SDS-PAGE before being transferred to PVDF membrane. Membranes were blocked in 10% normal goat serum for 1 h at room temperature before being placed into primary antibody for overnight incubation at 4°C. The following primary antibodies were used: mouse anti-β-actin (1:1000; Cell Signaling Technology, RRID: AB_2242334 ) and mouse anti-hnRNP A1 (1:1000; Millipore, RRID: AB_10562650 ). Membranes were washed and incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000; Bio-Rad, RRID: AB_11125547 ). Membranes were developed using Clarity Western ECL substrate (Bio-Rad) and visualized using the Bio-Rad ChemiDoc system. Three biological replicates of siNEG and siA1 transfected Neuro-2a cells were harvested and run for Western blotting. Blots were analyzed in ImageJ (RRID: SCR_003070 ) by densitometry and normalized to β-actin signal.
Cytobuster
Manufactured by Merck Group
Sourced in Germany, United States
CytoBuster is a laboratory instrument designed for cell lysis and protein extraction. It utilizes mechanical disruption to efficiently break down cells and release their intracellular contents, including proteins, enzymes, and other biomolecules. The CytoBuster is a versatile tool for researchers working in the fields of molecular biology, biochemistry, and proteomics.
Automatically generated - may contain errors
Lab products found in correlation
Bolt 10 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary anti mouse or anti rabbit igg, by Merck Group (2 mentions)
Immobilon ecl hrp substrate, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Phosstop, by Roche (2 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg secondary antibody, by Bio-Rad (1 mentions)
Mouse anti hnrnp a1, by Merck Group (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Sw60 rotor, by Beckman Coulter (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
13 protocols using cytobuster
1
Western Blot Analysis of hnRNP A1 in Neuro-2a Cells
2
Liver Protein Extraction Protocol
3
Budding of Virus-Like Particles Quantification
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Budding of VLPs into cell supernatants was detected by Western blot analyses. WT/mutant MeV-M bearing a FLAG-tag and NiV-M bearing a hemagglutinin-tag were cloned into pCMV and pcDNA3.1, respectively, and transfected into cells using TrasnIT-LT1 transfection reagent (Mirus). VLPs were harvested 24 hours after transfection. Cell culture medium was spun down at 3500 rpm for 20 min to pellet any cells out of the media. The cleared supernatants were then ultracentrifuged at 30,000 rpm with an SW-60 rotor (Beckman) for 2 hours through a 20% (w/v) sucrose cushion, 50 mM tris (pH 7.4), and 100 mM NaCl. Pelleted VLPs were resuspended in 1× NuPAGE LDS sample buffer (Thermo Fisher Scientific). Cell lysates were collected by washing cells twice with PBS followed by lysis in CytoBuster (Millipore). VLPs and cell lysates were electrophoresed on SDS denaturing gels, transferred onto polyvinylidene difluoride Immobilon-P transfer membranes (Millipore), and probed with an anti-Flag or anti–NiV-M primary antibody. The relative intensities of the bands were quantified by densitometry with a ChemiDoc MP imaging system (Bio-Rad) and ImageJ. The budding index was defined as the amount of MeV-M or NiV-M in the VLPs divided by the amount in the cell lysate across each independent experiment and normalized to the average WT MeV-M or NiV-M.
4
Western Blot Analysis of Signaling Pathways
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Cells were seeded at a density of 1 × 106 cells/plate on 60 mm cell culture dish overnight. Cells were incubated with M5 or PG102 at designated concentrations for 30 min and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents in the cell lysates were determined by bicinchoninic acid (BCA) assay kit and after reconstituting in sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane (Merck) and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 h to block nonspecific binding. The membrane was then incubated with antibodies specific for phospho-STAT3 (#9134, #9145), STAT1 (#9167), p65 (#3033), and STAT3 (#4904), STAT1 (#9172), p65 (#8242), IκB-α (#9242) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (A5441, Sigma-Aldrich) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich) at room temperature for 1 h. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure on autoradiography film.
5
Western Blot Analysis of Protein Extracts
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Proteins were extracted in hot Laemmli sample buffer and subjected to Western blot analysis. 30 μg total protein extracts were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal AR (AR441, NeoMarkers-Lab Vision Corporation, Fremont, CA, USA), goat polyclonal anti-β-Actin (Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with mouse monoclonal anti-CIP2A (Santa Cruz Biotechnology, Santa Cruz, CA). For primary PC cells CytoBuster (Merck Millipore) buffer was used to lyse freshly cultured cells to extract proteins.
6
Quantification of aortic tissue proteins
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Aortic tissues and 20 μM montelukast-induced macrophages were homogenized using an ultrasonic disintegrator (Sonics & Materials, Inc., Newtown, CT, USA) in protein extraction buffer (CytoBuster; Novagen, Merck KGaA, Darmstadt, Germany) with 20 mM of ethylenediaminetetraacetic acid (Dojindo, Kumamoto, Japan) and 1 mM of phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). Lysate protein concentration was measured using the Qubit® Protein Assay Kit and Qubit® 2.0 fluorometer (Thermo Fisher Scientific). An equal concentration of total protein was applied to each assay kit and detected according to the manufacturer's protocol for each ELISA kit (TIMP-1 and TGF-β1: R&D Systems, Minneapolis, MN, USA; TIMP-2: RayBiotech, Norcross, GA, USA; IGF-1: Mediagnost, Reutlingen, Germany; IL-1β, IL-6, TNF-α, and MCP-1: Bender MedSystems, Vienna, Austria; IL-10: Thermo Fisher, Waltham, MA, USA) [12 (link)]. Seven samples were analyzed per group.
7
Affinity Purification of Recombinant IRE1α Protein
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Cell pellets were thawed, resuspended, and lysed in ‘cytobuster’ (EMD Millipore) containing protease (EDTA-free) and phosphatase inhibitor cocktails (Pierce) and 30 mM imidazole, pH 8.0. The resulting cell lysates were clarified and affinity purified on HisTrapFF nickel columns (GE Healthcare) using an AKTA FPLC (GE Healthcare, Marlborough, MA, USA). Bound protein was eluted in a linear gradient of elution buffer (50 mM sodium phosphate buffer, pH 7.2, 500 mM NaCl, 500 mM imidazole). Peak fractions were run on SDS-PAGE gels and analyzed by Coomassie staining. IRE1α containing fractions were pooled and concentrated in Amicon Ultra 30K cut off concentrators (EMD Millipore) and buffer was exchanged into 25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT, 5% glycerol. Protein concentration was determined by Bradford assay. Protein was aliquoted and stored at −80 °C, with additional glycerol (25% final) added for longer term storage. This expression and purification approach has been successfully used to study IRE1α kinase activity in vitro [58 (link),60 (link),72 (link)].
8
Protein Expression Analysis in HaCaT Cells Treated with PG102
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HaCaT cells were seeded at a density of 4.5 × 105 cells/well onto 6-well plates overnight. Cells were then treated with PG102 at designated concentrations for 48 hours, and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents were measured by the BCA assay kit (Invitrogen), and after reconstitution in a sample buffer, 20 micrograms of protein samples was subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane, and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 hour to block nonspecific binding. The membrane was then incubated with antibodies specific to IL-37, phopho-Smad3 (1 : 1000; Abcam), Smad3, phospho-ERK, ERK (1 : 1000; Cell Signaling Technology, Danvers, MA), and β-actin (1 : 5000; Sigma-Aldrich) overnight at 4°C followed by incubation with a horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1 : 100,000; Sigma-Aldrich) at room temperature for 1 hour. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure to autoradiography film.
9
Asp Protein Detection Using Urea-Based Buffer
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Consistent with a recent study (Rujano et al., 2013 (link)), we also failed to detect the Asp protein using a standard procedure with SDS sample buffer. In this study, we prepared the sample in two ways. In Fig. S1 B, we treated cells with a urea-containing solution (62.5-mM Tris, pH 6.8, 10% glycerol, 2% SDS, 4-M urea, and 2-mercaptoethanol) at room temperature for >1 h. In Fig. S1 A, we first treated cells with the Cytobuster (EMD Millipore) solution that also contained benzonase, DTT, and protein inhibitors for 30 min on ice, followed by addition of the urea-containing solution. Rabbit polyclonal anti-Asp antibody was generated by using recombinant Asp [1–500 aa] as the antigen, and the crude serum was used for immunoblotting after a 1:500 dilution. Rabbit polyclonal anti-Ncd antibody was a gift from J. Scholey (University of California, Davis, Davis, CA; Morales-Mulia and Scholey, 2005 (link)).
10
Western Blot Analysis of TLR7, TLR8, and COX-2
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Proteins were extracted from tissue samples (250 μg) using lysis buffer CytoBuster (Merck, Darmstadt, Germany) and QIAshredder (Qiagen, Hilden, Germany). Normal tissue (protein lysate) was purchased from BioChain Institute Inc. (Hayward, CA, USA). Protein samples (50 μg) were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). Blots were probed with antibodies to TLR7 (ProSci), TLR8 (ProSci), β-actin (Santa Cruz Biotechnology) and COX-2 (Santa Cruz Biotechnology and Novus Biologicals LLC, Littleton, CO, USA). Anti-mouse IgG and anti-rabbit IgG secondary antibodies were obtained from Amersham (Braunschweig, Germany) and anti-goat IgG was purchased from Santa Cruz Biotechnology.
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