Protein was isolated in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phosphatase inhibitor cocktail 3 (p0044, Sigma‐Aldrich), 1x cOmplete protease inhibitor cocktail (11 873 580 001, Roche), and 15 mM sodium orthovanadate (S6508, Sigma‐Aldrich). Protein concentration was determined with the DC protein assay kit. Equal amounts of protein were separated by SDS‐PAGE and proteins were transferred to PVDF membrane. For detection of specific proteins, the following antibodies were used: polyclonal anti‐cathepsin D IgG (1:1000; sc‐10 725, Santa Cruz), monoclonal anti‐Caspase 3 (1:1000; 9664, Cell Signalling) and monoclonal anti‐GAPDH IgG (1:30.000; 10R‐G109A, Fitzgerald). After washing, blots were incubated with polyclonal goat anti‐rabbit IgG‐HRP (1:2000; P0448, Dako), and polyclonal rabbit anti‐mouse IgG‐HRP (1:2000; P0260, Dako). Signals were detected visualized with enhanced chemiluminescence (ECL; NEL120001EA, PerkinElmer) and densitometry has been analysed with ImageQuant LAS 4000 (GE Healthcare). Cathepsin D signals were normalized to respective GAPDH levels.
P0260
Manufactured by Agilent Technologies
Sourced in Denmark, United States, Germany, Belgium
The P0260 is a laboratory equipment product manufactured by Agilent Technologies. It is a precision instrument designed to perform specific functions within a controlled laboratory environment. The core function of the P0260 is to provide accurate and reliable measurements or analysis, as required by the user's intended application. No further details about the intended use or interpretation of the product's capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
P0448, by Agilent Technologies (17 mentions)
P0217, by Agilent Technologies (17 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
P0399, by Agilent Technologies (6 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
P0160, by Agilent Technologies (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Azure c600 imager, by Azure Biosystems (3 mentions)
Western bright chemiluminescence reagent, by Cytiva (3 mentions)
Western lightning plus ecl, by PerkinElmer (3 mentions)
Sc 365062, by Santa Cruz Biotechnology (3 mentions)
A5441, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Ab14745, by Abcam (2 mentions)
Ab14705, by Abcam (2 mentions)
Ab110242, by Abcam (2 mentions)
111 protocols using p0260
1
Cathepsin D and Caspase 3 Immunoblotting Assay
2
Citrullination of Fibrinogen Assay
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As described11 (link), Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 1 µg/ml fibrinogen (Calbiochem, Darmstadt, Germany). Wells were washed thrice and blocked in Tris-buffered saline (TBS) buffer containing 0.05% Tween-20, pH 7.4. Hereafter, the wells were incubated for 3 h at room temperature (RT) with PAD-containing samples with or without GSH, H2O2, and EDTA, as stated in figure legends. Purified leukocytes were applied in RPMI 1640 containing 5% AB serum and, when relevant, 15 nM PMA, 25 µM DPI, which inhibits NOX2 and other flavoenzymes by interacting with the flavin part30 (link), or H2O2 at various concentrations. After three washes in washing buffer (PBS, 0.05% Tween-20, pH 7.4), mouse anti-citrullinated fibrinogen antibody (0.5 µg/ml) and hereafter horse radish peroxidase-conjugated polyclonal rabbit-anti mouse immunoglobulin antibodies (P0260, Dako, Glostrup, Denmark) were added and developed as described11 (link). Optical density (OD) was measured at 490–650 nm using the SPECTROstar nano Microplate Reader (BMG Labtech, Ortenberg, Germany). Data were processed using MARS software (BMG Labtech).
3
Western Blot Analysis of PbNK65 Protein
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20 µg PbNK65 WT protein extract from asexual blood stages were separated by SDS-PAGE (4–12% Bis-Tris gels; BOLT mini gel system; Thermo Fisher Scientific) and transferred onto a polyvinylidene fluoride membrane (iBLOT system; Thermo Fisher Scientific). Sera from uninfected, WT, or PbNK65-hrfΔ1 mice were added (1:1,000 dilution) and incubated overnight at 4°C. After washing the membrane with PBS + Tween 20 (Sigma-Aldrich), polyclonal anti–mouse IgGs (1:20,000; P0260; Dako) were added, and specific bands were visualized with the SuperSignal West Pico kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
4
ACE2 Binding Assay for Coronavirus S1 Proteins
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The ability of the HCoV-NL63 S1-mFc and S1-B-mFc chimeric proteins to bind the ACE2-hFc receptor was evaluated with an ELISA-based assay. 100 μl of hACE2-hFc (20 μg/ml, diluted in PBS) was coated on a 96-well MaxiSorb plate overnight at 4 °C. Nonspecific binding sites were subsequently blocked with a 3% (w/v) solution of bovine serum albumin in PBS. Plates were washed with washing buffer (PBS with 0.05% Tween 20) and subsequently incubated with serially diluted S1-mFc proteins (starting with equimolar concentrations) for 1 h at room temperature, after which plates were washed three times with washing buffer. mFc-tagged S1 proteins were detected with HRP-conjugated polyclonal rabbit-anti-mouse immunoglobulins (1:2,000 dilution in PBS with 0.1% BSA; DAKO, P0260), and a colorimetric reaction was produced after incubation with tetramethylbenzidine substrate (BioFX). The optical density (OD) was subsequently measured at 450 nm with an ELISA reader (EL-808, BioTEK). Background (signal from HRP-conjugated anti-mFc antibody alone) was subtracted from the OD450nm values. The mFc-tagged SARS-CoV S1 subunit was used as a positive control, whereas the mFc-tagged HCoV-NL63 S1 domain 0 (HCoV-NL63 S1-0-mFc) and PEDV S1 subunit (PEDV S1-mFc), both of which do not bind ACE2, were used as negative controls.
5
Western Blot Analysis of Skeletal Muscle
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Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.30 (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.
6
Quantification of IDE and Aβ Levels
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For Western Blot analysis of IDE and β-actin protein levels, samples were prepared as described above, adjusted to equal protein amounts and loaded on 10–20% tris-tricine-gradient gels (Anamed Elektrophorese, Groß-Bieberau/Rodau, Germany). For measuring total Aβ degradation, the cell culture supernatant containing remaining human Aβ40 was also separated in tris-tricine-gradient gels. After transferring proteins onto nitrocellulose membranes, IDE, β-actin and human Aβ40 were detected with the primary antibodies ST1120 (1:2000), A5441 (1:5000) and W02 (1 μg/mL), all purchased from Merck, respectively. HRP-coupled antibodies W401 (anti-rabbit, 1:5000) (Promega, Mannheim, Germany) and P0260 (anti-mouse, 1:5000) (Dako, Hamburg, Germany) were utilized as secondary antibodies. Signal detection was performed with the enhanced chemiluminescence (ECL-) method (Perkin Elmer, Rodgau-Jügesheim, Germany) and densitometrical quantification of band intensity was carried out with Image Gauge software (Version 3.45) (Fujifilm). Total proteins were detected using Ponceau S staining as described in [52 (link)] before immunodetection.
7
Evaluating Apoptotic Pathways in MDA-MB-231 Cells
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Upon treatment with Dox@IONs-POES or Elli@IONs-PVP (5 μg/mL of each topo II poison, 6 hrs), total cellular proteins from MDA-MB-231 cells were extracted with 100 µL of RIPA buffer containing protease inhibitor cocktail. After electrophoresis, the proteins were electrotransferred onto the Immuno-Blot® PVDF membrane (Bio-Rad, Hercules, CA, USA) and blocked with 10% (w/v) skim milk powder for 1 hr at 37°C. Membranes were incubated with primary mouse anti-GAPDH (1:700), mouse anti-β-actin (1:700), mouse anti-Bcl-2 (1:200), mouse anti-p53 (1:250), mouse anti-MT1-1/2 (1:200) and mouse anti-MT-3 (1:200). After washing, membranes were incubated with relevant horseradish peroxidase-labeled secondary antibody (p0260, 1:5,000, Dako, Glostrup, Denmark) for 1 hr at 20°C. Signals were developed using Clarity Western ECL Blotting Substrate (Bio-Rad) and blots were visualized using Azure c600 imager (Azure Biosystems).
8
Immunostaining of Myosin Heavy Chain Isoforms
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Serial transverse 10 mm thick cryosections were marked with three monoclonal antibodies (BA-D5, BF-F3, and SC71) purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ, Braunschweig, Germany and a fourth (6H1) sourced from the Developmental Studies Hybridoma Bank, Iowa City, USA[24 (link),25 (link)]. The antibodies were immunoreactive to specific myosin heavy chain isoforms: BA-D5 antibodies for myosin heavy chain-1; BF-F3 for myosin heavy chain-2b; SC71 for myosin heavy chain-2a; and 6H1 for myosin heavy chain-2x/d. All antibody applications were at 1:100 dilutions, except for BF-F3 at 1:30. Myosin heavy chain isoforms 1, 2a, and 2b were revealed by a horseradish peroxidase-conjugated secondary antibody P0260 (Dako, Glostrup, Denmark), while 2x/d was revealed by a Polymer Detection System (Novolink, Leica Biosystems, Newcastle, UK). The expression of myosin heavy chain isoforms on serial sections by the indirect immunoperoxidase method was used for muscle fiber phenotyping, while the specificity of immunostaining was confirmed by lack of immunoreactivity in sections incubated without primary antibodies [26 (link)-28 (link)].
9
Immunoblotting Analysis of Alpha-Synuclein
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Primary antibodies: α-syn monoclonal mouse-anti-α-syn (SYN-1, BD Biosciences, catalog no. 610787), polyclonal rabbit-anti-α-syn (ASY147 (link)), rabbit-anti-α-syn (phospho S129) (abcam, catalog no. ab51253), rabbit-anti-α-syn (phospho S129) (Cell Signaling, D1R1R, mAb #23,706), MJFR-14-6-4-2 (MJF14) (abcam, catalog no. 209538, rabbit monoclonal antibody, generated against full length α-syn protein filament), anti-α-tubulin (T9026 Sigma-Aldrich Co.) and anti-Tyrosine Hydroxylase (GeneTex, GTX113016). Secondary antibodies: horseradish peroxidase (HRP)-conjugated polyclonal rabbit-anti-mouse (Dako, P0260), HRP-conjugated polyclonal swine-anti-rabbit (Dako, P0217).
10
Duodenal Protein Expression Analysis
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