Purified EVs were lysed with 2× sodium dodecyl sulfate (SDS) sample buffer [100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol]. Total proteins were separated by SDS-PAGE, and then the following primary antibodies were used: anti-CD63 (mouse monoclonal, SHI-EXO-M02, 1:500, COSMO BIO Co. Ltd., Tokyo, Japan), anti-CD9 (mouse monoclonal, SHI-EXO-M01, 1:500, COSMO BIO Co. Ltd), anti-CD81 (mouse monoclonal, MA5-13548, 1:100, Thermo Fisher Scientific), anti-flotillin 1 (rabbit monoclonal, ab133497, 1:10,000, abcam). Subsequently, the following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse IgG (NA931-1ML, 1:4,000, GE Healthcare UK Ltd, England), anti-rabbit IgG-HRP (NA934-1ML, 1:1,000, GE Healthcare UK Ltd). Protein bands were quantified by densitometry using Image Quant LAS 4000 (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England).
Na931 1ml
Manufactured by GE Healthcare
Sourced in United Kingdom
The NA931-1ML is a lab equipment product from GE Healthcare. It is designed for precise liquid handling tasks in laboratory settings. The core function of this product is to accurately measure and dispense small volumes of liquids with high repeatability.
Automatically generated - may contain errors
Lab products found in correlation
Na934 1ml, by GE Healthcare (5 mentions)
Na934, by GE Healthcare (2 mentions)
Qubit protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Roti load reducing buffer, by Carl Roth (2 mentions)
Phosstop, by Roche (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab133497, by Abcam (1 mentions)
Shi exo m01, by Cosmo Bio (1 mentions)
Ma5 13548, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ha y 11, by Santa Cruz Biotechnology (1 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
H4k16ac, by Merck Group (1 mentions)
Mouse anti crisprcas9 antibody ab191468, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Fusion solo s, by Vilber (1 mentions)
Sting, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
11 protocols using na931 1ml
1
Extracellular Vesicle Protein Analysis
2
Heterologous expression of PKD proteins
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Human PKD2L1 cDNA (accession #: NM_016112) was inserted into vector pCHGF61 (link) for efficient expression in Xenopus laevis oocyte. Flag tag was then added before the N-terminus of the PKD2L1 for immunodetection. HA-tagged human PKD2 (NM_000297) plasmid was constructed as we previously reported5 (link). Mutagenesis was carried out using QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, La Jolla, CA) and confirmed by sequencing. Sequences of primers used in this study are included in Supplementary Table 2 . Rabbit FLAG (D-8, 1:2000), HA (Y-11, 1:2000) and mouse β-actin (C-4, 1:4000) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) for immunoblotting. Secondary antibodies against rabbit (NA934-1ML, 1:2000) or mouse (NA931-1ML, 1:2000) lgG were from GE Healthcare (Waukesha, WI).
3
Whole-cell Extraction and Western Blot Analysis
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For whole-cell extracts, cells were lysed in 50 mM NaCl, 1.0% IGEPAL CA-630, and 50 mM tris-HCl (pH 8.0). Buffer was supplemented with cOmplete (Roche, #4693132001) and PhosSTOP (Roche, #4906845001). Protein concentration was determined by Qubit protein assay reagent (Thermo Fisher Scientific, #Q33212). Samples were denatured at 95°C for 5 min in a Roti-Load reducing buffer (Carl Roth, #K929.1) before SDS–polyacrylamide gel electrophoresis (PAGE) using NuPAGE bis-tris gels and then transferred to 0.45 μM polyvinylidene difluoride membranes. Membranes were blocked for 30 min with 5% milk in PBS with 0.3% Tween (PBST). Membranes were washed twice with 0.3% PBST. The membrane was incubated with primary antibodies against MOF (1:1000; Bethyl, #A300-992A), actin (1:10000; Sigma-Aldrich, #A2066), H3 (1:5000; Active Motif, #39763), and H4K16ac (1:2000; Millipore, #07-329) diluted in PBS–5% BSA. The membrane was then incubated with PBS–5% BSA containing horseradish peroxidase–conjugated anti-mouse (GE Healthcare, #NA931-1ML) or anti-rabbit (GE Healthcare, #NA934) (1:10,000).
4
Quantifying Cas9 Protein Expression
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Cell pellets (1 × 106 cells) were acquired at 12, 24, 48, 72 h and 7 days post electroporation with coBE3 mRNA (50 μg/mL). Total protein was quantified by a bicinchoninic acid (BCA) assay. Western blot was run using 25 μg total protein per sample. 16.5 ng Alt-R® S.p. HiFi Cas9 Nuclease V3 (1081061, IDT, Leuven, Belgium) was used as positive control. Membrane was blotted with mouse anti-CRISPRCas9 antibody (ab191468, abcam, Cambridge, UK) at a 1:1000 dilution in 3% milk overnight at 4 °C before incubation with secondary HRP-linked sheep anti mouse (NA931-1ML, GE Healthcare Life Sciences, Buckinghamshire, UK) at a 1:3000 dilution in 5% milk for 1 h at room temperature. Protein was visualised by chemiluminescence using Pierce ECL western blotting substrate (32106, ThermoFisher Scientific).
5
Comprehensive Western Blotting Protocol
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For western blotting, cells were lysed in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20 and 10 mM β-glycerophosphate) containing 1% protease inhibitor cocktail (Nacalai Tesque, 25955-11). The protein concentration was determined using the DC Protein Assay (Bio-Rad), and the proteins were separated via SDS-PAGE and then transferred to PVDF membranes (EMD Millipore). After blocking with 5% milk, membranes were probed with primary antibodies targeting HRas (Santa Cruz, sc-29), p16 (IBL, 11104), STING (Cell Signaling Technology, 13647), cGAS (Cell Signaling Technology, 15102), Lamin B1 (Abcam, ab16048), DP1 (Abcam, ab11834), α-tubulin (Sigma-Aldrich, T9026), RNaseH2A (PROTEINTECH, 16132-1-AP), E2F3 (Santa Cruz, sc-878), mini-AID-tag (MBL, M214-3), β-actin (Santa Cruz, sc-47778) and GAPDH (PROTEINTECH, 60004-1-Ig). The membranes were incubated with a mouse (GE Healthcare, NA931-1ML) or rabbit secondary antibodies (GE Healthcare, NA934-1ML), visualised using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096) and detected using FUSION SOLO S (Vilber Lourmat).
6
Protein Extraction and Western Blotting
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For protein extraction, cultured cells were homogenized with 200 µl of RIPA lysis buffer (Thermo Fisher Scientific), 2 µl of proteinase inhibitor (Nacalai Tesque), and 2 µl of phosphatase inhibitor cocktail (Nacalai Tesque). After centrifuging (15,000 rpm, 10 min), supernatants were collected. Total protein was quantified using Protein Assay CCB Solution (Nacalai Tesque). Twenty micrograms of protein was fractionated with sample buffer solution with reducing reagent for SDS‐PAGE (Nacalai Tesque) on acrylamide gel (Mini‐Protean TGX Precast Gel, Bio‐Rad) and electro‐transferred to nitrocellulose membranes. After blocking with 5% skim milk for 1 h, the membranes were probed with antibodies against SNCG (1:200 dilution; 1H10D2, Santa Cruz Biotechnology) or β‐actin (1:500 dilution; C4, Santa Cruz Biotechnology) overnight in a cold room. Peroxidase‐conjugated anti‐mouse antibody (1:5000 dilution; NA931‐1ML, GE Healthcare Life Sciences) was used as a secondary antibody and signals were enhanced by highly sensitive detection substrate (Chemi‐Lumi One Super, Nacalai Tesque).
7
Multiplexed Immunofluorescence Imaging of LCN-2 and Cytokeratin
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4-µm sections of the recipient block were mounted onto slides, subjected to antigen retrieval, and stained with an antibody combination consisting of LCN-2 (R&D, MAB1757) and pan-cytokeratin (panCTK) (Abcam, ab7753) according to manufacturer´s instructions using the Opal™ 4-Color Fluorescent IHC Kit (Perkin-Elmer). DAPI was used for nuclear visualization. Horseradish peroxidase (HRP) coupled anti-mouse (GE Healthcare, NA931.1 ML) or anti-rat (GE Healthcare, NA935-1 ML) secondary antibodies was used for detection. Images were acquired using the Vectra automated imaging system and analysis was performed using ImageJ. Specifically, the background was substracted using the rolling circle function and the channels were demixed during this process (the rolling circle radius was set to at least the size of the largest object that is not part of the background which was determined a priori). Thresholds were set semi-automatically for the pre-processed images. Under- or overexposed images were discarded from further analysis. For quantitative analysis, the images were then converted to binary images and the signal distribution was determined for each color individually.
8
Western Blot Analysis of Cellular Proteins
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As described in (37 (link)), for whole-cell extracts, cells were lysed in 50 mM NaCl, 1.0% IGEPAL CA-630, and 50 mM tris-HCl (pH 8.0). Buffer was supplemented with cOmplete (Roche, #4693132001) and PhosSTOP (Roche, #4906845001). Protein concentration was determined by a Qubit protein assay reagent (Thermo Fisher Scientific, #Q33212). Samples were denatured at 95°C for 5 min in a Roti-Load reducing buffer (Carl Roth, #K929.1) before SDS–polyacrylamide gel electrophoresis using NuPAGE bis-tris gels and then transferred to 0.45 μM polyvinylidene difluoride membranes. Membranes were blocked for 30 min with 5% milk in PBS with 0.3% Tween 20 (PBST 3%). Membranes were washed twice with PBST 3%. The membrane was incubated with primary antibodies against MOF (1:1000; Bethyl, #A300-992A), actin (1:10,000; Sigma-Aldrich, #A2066), H3 (1:5000; Active Motif, #39763), p16INKA (1:1000; Abcam, #ab54210), p19INKD (1:1000; Abcam, #ab80), and H4K16ac (1:2000; Millipore, #07-32) diluted in PBS–5% BSA. The membrane was then incubated with PBS–5% BSA containing horseradish peroxidase–conjugated anti-mouse (GE Healthcare, #NA931-1ML) or anti-rabbit (GE Healthcare, #NA934) (1:10,000).
9
Western Blot Protein Analysis Protocol
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Protein samples were run on 4–20% Tris-Glycine SDS gradient gels and transferred to PVDF membranes using the semi-dry Trans-Blot Turbo system (Bio-Rad). In all, 5% weight/volume milk in PBST was used for blocking and all antibody dilutions. Primary antibodies were incubated on the blots overnight at 4 °C, while horseradish peroxidase-coupled secondary antibodies were on blots for 1 h at room temperature. Clarity Western ECL Substrate (Bio-Rad, 1705060) was used for development, and blots were imaged on a ChemiDoc MP (Bio-Rad). Primary antibodies used: anti-RPL10A (Abcam ab174318, 1:1000 dilution), anti-RPL10A (Santa Cruz Biotechnology sc-100827, 1:1000 dilution), anti-RPL11 (Abcam ab79352, 1:1000 dilution), anti-RPL34 (Abcam ab129394, 1:500 dilution), anti-RPL23A (Bethyl A303-932A-M, 1:2000 dilution), anti-RPL22 (ProteinTech 250021AP), anti-RPS25 (Sigma HPA031801, 1:250 dilution), anti-RPS5 (Abcam ab58345, 1:1000 dilution), anti-GAPDH (Invitrogen AM4300, 1:2000 dilution), anti-β-actin (Cell Signaling 3700 S, 1:2000 dilution). Secondary antibodies used: donkey anti-mouse (GE Healthcare NA931-1ML, 1:5000 dilution) or donkey anti-rabbit (GE Healthcare NA934-1ML, 1:5000 dilution).
10
Western Blot Analysis of ABC Transporters
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Samples were prepared, separated and transferred to PVDF membranes as described previously (Aoshima et al., 2018) . Membranes were either incubated with anti-ABCB1 antibody (#ab170904, 1:2,500, abcam, Cambridge, UK), anti-ABCG2 antibody (#ab3380, 1:1,000, abcam), anti-ATM antibody (#NB100-104, 1:1,000, Novus biologicals, CO, USA), anti-cleaved caspase-3 (Asp175) antibody (#9661, 1:1,000, Cell signaling, MA, USA) (Penzo-Méndez et al. 2015) or anti-actin antibody clone C4 (#MAB1501, 1:10,000, Merck Millipore) for overnight at 4°C. After washing with Tris-buffered saline containing 0.05% Tween 20 (TBST), membranes were incubated with ECL rabbit IgG HRP-linked whole Ab (#NA934-1ML, 1:10,000, GE Healthcare, IL, USA) or ECL mouse IgG HRPlinked whole Ab (#NA931-1ML, 1:10,000, GE Healthcare) for 1 hour at RT. After washing with TBST, signals were developed with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) and detected by ImageQuant LAS 4000 mini (GE Healthcare). Signal intensities were obtained by ImageJ software (Rasband, 1997 (Rasband, -2018;; Schneider et al., 2012) . Expression levels of ATM and cleaved caspase-3 were normalized with actin expression levels.
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