Equal amounts of protein lysates were mixed with NuPAGE LDS Sample Buffer. Proteins were separated using the SDS-PAGE method at 150 V for 1 h and transferred at 30 V for 1 h at 4°C on the polyvinylidene fluoride membrane (IPVH00010; Millipore) with the following block in 5% skim milk, diluted in Tris-buffered saline with 0.05% Tween-20 (TBST; P7949; Sigma). Blots were incubated for 18 h at 4°C with one of the following primary antibodies: annexin A1 (AF3770; R&D Systems), actin (sc-47778; Santa Cruz Biotechnology), or GAPDH (D16H11; Cell Signaling Technology). Membranes were washed thrice in TBST, followed by the addition of horseradish peroxidase-conjugated secondary antibodies (all Amersham), and incubation for 1 h at room temperature. Membranes were washed thrice in TBST, and protein bands were visualized with enhanced chemiluminescence using an X-ray detector.
P7949
Manufactured by Merck Group
P7949 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of this product is to perform specific tasks within the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Oct compound, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Nupage novex 3 8 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
A7906, by Merck Group (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Celldiscoverer 7, by Zeiss (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
A3311, by Merck Group (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Superdex 75 increase, by GE Healthcare (1 mentions)
Dithiothreitol (dtt), by neoFroxx (1 mentions)
Odyssey fc, by LI COR (1 mentions)
14 protocols using p7949
1
Western Blot Analysis of Protein Expression
2
Whole-Cell Protein Extraction and Western Blotting
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To prepare whole-cell extracts, cells were washed once in 1× PBS, harvested by trypsinisation, washed in 1× PBS and re-suspended in SDS-PAGE loading buffer (0.16 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 100 mM DTT). Samples were sonicated and heated at 70 °C for 10 min. Equal amounts of protein (20–80 µg) were analysed by gel electrophoresis followed by Western blotting. NuPAGE-Novex 10% Bis-Tris (MOPS buffer, Life Technologies) and NuPAGE-Novex 3–8% Tris-Acetate gels (Life Technologies) were run according to the manufacturer’s instructions. Proteins were transferred to 0.2 μM nitrocellulose membranes (GE Healthcare) using semi-dry transfer in transfer buffer (Life Technologies, NP00061) with 10% methanol. Membranes were blocked in 5% skimmed milk in PBS-Tween (PBST; 0.05% Tween 20 [Sigma-Aldrich, P7949] in PBS) then incubated overnight at 4 °C in primary antibody diluted in 2% bovine serum albumin (BSA; Sigma-Aldrich, A7906) in PBST. After washes with PBST, the membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, DAKO, P0447 and P0448 respectively) at room temperature for 1 h. The membranes were washed with PBST and developed by enhanced chemiluminescence (ECL; GE and Millipore).
3
Cytoskeletal and Nuclear Imaging of HDF Cells
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Immunofluorescence
of nuclei and the cytoskeleton of the HDF cells were carried out on
days 4 and 7. First, the cells were washed with DPBS and then fixed
in 4% PFA for 15 min, following triple DPBS wash. Then, the cells
were incubated for 1 h with a solution of 0.001% Tween 20 (P7949,
Sigma-Aldrich) and 1:100 Alexa Fluor 488 phalloidin (A12379, Thermo
Fisher Scientific) in DPBS. Wells were subsequently rinsed three times
with DPBS and incubated for 5 min with 1:10,000 Hoechst (62249, Thermo
Fisher Scientific) in DPBS. Imaging was performed with a confocal
microscope (Zeiss Cell Discoverer 7).
of nuclei and the cytoskeleton of the HDF cells were carried out on
days 4 and 7. First, the cells were washed with DPBS and then fixed
in 4% PFA for 15 min, following triple DPBS wash. Then, the cells
were incubated for 1 h with a solution of 0.001% Tween 20 (P7949,
Sigma-Aldrich) and 1:100 Alexa Fluor 488 phalloidin (A12379, Thermo
Fisher Scientific) in DPBS. Wells were subsequently rinsed three times
with DPBS and incubated for 5 min with 1:10,000 Hoechst (62249, Thermo
Fisher Scientific) in DPBS. Imaging was performed with a confocal
microscope (Zeiss Cell Discoverer 7).
4
Coating PMMA Microbeads with Cdh1 Protein
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PMMA microbeads (Microparticles, PMMA-R-B375) 36 μm in diameter, washed in 0.01% Tween20 (Sigma, P-7949) in DPBS (DPBS-T), were used to establish contact with 8-cell stage blastomeres. To coat microbeads with Cdh1, recombinant mouse Cdh1-Fc chimera protein (RnDsystems, 748-EC-050) was reconstituted at 100 μg/ml in sterile DPBS with Ca2+ and Mg2+ (Gibco, 14040-091). Protein A-coated PMMA microbeads (Microparticles, PMMA-Protein A-S2976B) 36 μm in diameter, were washed in 0.01% DPBS-T and incubated in 0.8 μg/ml Cdh1 solution at 1600 beads/ml for 90 min at 4°C with 1400 rpm mixing (Thermomixer, Eppendorf). After washing with 0.01% DPBS-T, microbeads were incubated in 1% heat-inactivated BSA (80°C, 10 min; Sigma, A3311) overnight at 4°C to block non-coated sites.
5
Ark2C-Ubiquitin Interaction Analysis
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GST pull-down assays were performed by mixing GST-Ark2C proteins immobilised on Glutathione SepharoseTM resin (GE Healthcare) with ubiquitin. Samples were made up to 200 μL in a buffer containing PBS pH 7.4, 0.2% Tween® 20 (P7949, Sigma-Aldrich), and 2 mM DTT (1114GR005, BioFroxx), and incubated on a tube rotator at 4 °C for 30 min. Samples were then washed four times with PBS before being mixed with reducing 2× SDS sample buffer and resolved by SDS-PAGE. The pulldowns containing Cy3-labelled ubiquitin were imaged using an Odyssey® Fc (LI-COR Biosciences) with a 600 nm filter prior to Coomassie staining.
Analytical SEC was performed using a 10/300 Superdex 75 increase (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl buffer. For each run, 100 µL of 200 µM of purified Ark2C variant, ubiquitin, or a mixture of the two proteins was resolved. For mixtures, samples were incubated overnight prior to separation. The recovered fractions were analysed by SDS-PAGE.
Analytical SEC was performed using a 10/300 Superdex 75 increase (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl buffer. For each run, 100 µL of 200 µM of purified Ark2C variant, ubiquitin, or a mixture of the two proteins was resolved. For mixtures, samples were incubated overnight prior to separation. The recovered fractions were analysed by SDS-PAGE.
6
Immunohistochemistry of Hair Follicles
7
Immunostaining of Pluripotent Stem Cells
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The immunostaining assay was conducted as follows.
Transgenic hESCs Royan H5 and H6 were fixed with 5% paraformaldehyde (Sigma-Aldrich,
P6148) for 10 minutes, after which their membranes
were permeabilized by 0.3% Triton X-100
(Sigma-Aldrich, T8532) and blocked with 10%
host serum in 1% bovine serum albumin (Sigma-
Aldrich, A3311). The cells were placed overnight
at 4˚C with the following primary antibodies:
mouse anti-SSEA4 (1:250) and mouse anti-OCT4
(1:250) diluted in blocking solution. Washing was
performed three times with 0.1% Tween 20 (Sigma-
Aldrich, P7949) in PBS-, and cells were incubated
at 37˚C with the following secondary antibodies-
goat anti-mouse fluorescein isothiosyanat
(FITC) conjugated (1:200, Santa Cruz Biotechnology,
sc-2010) and goat anti-mouse Dylight conjugated
(1:200, Santa Cruz Bio-technology, sc-2780)
for 45 minutes. Nuclei were counterstained with
4΄,6-diamidino-2-phenylindole (DAPI, 1:1000,
Sigma-Aldrich, D8417) and analyzed with a fluorescence
microscope (Olympus, IX71) (Fig .2 ).
Transgenic hESCs Royan H5 and H6 were fixed with 5% paraformaldehyde (Sigma-Aldrich,
P6148) for 10 minutes, after which their membranes
were permeabilized by 0.3% Triton X-100
(Sigma-Aldrich, T8532) and blocked with 10%
host serum in 1% bovine serum albumin (Sigma-
Aldrich, A3311). The cells were placed overnight
at 4˚C with the following primary antibodies:
mouse anti-SSEA4 (1:250) and mouse anti-OCT4
(1:250) diluted in blocking solution. Washing was
performed three times with 0.1% Tween 20 (Sigma-
Aldrich, P7949) in PBS-, and cells were incubated
at 37˚C with the following secondary antibodies-
goat anti-mouse fluorescein isothiosyanat
(FITC) conjugated (1:200, Santa Cruz Biotechnology,
sc-2010) and goat anti-mouse Dylight conjugated
(1:200, Santa Cruz Bio-technology, sc-2780)
for 45 minutes. Nuclei were counterstained with
4΄,6-diamidino-2-phenylindole (DAPI, 1:1000,
Sigma-Aldrich, D8417) and analyzed with a fluorescence
microscope (Olympus, IX71) (
8
Biotin-PrP Binding Interaction Assay
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Full‐length PrPC(23‐230) and PrPC(23‐111) protein were biotinylated by incubation with N‐hydroxy‐succinimide‐activated biotin and free biotin was removed. The biotin‐PrPC was immobilized on streptavidin‐coated BioLayer Interferometry (BLI) probes (Pall ForteBio, 18‐5019). Interaction with different concentrations of AZ59 in PBS in 0.05% Tween 20 (Sigma, P7949) was detected and analyzed by an OctetRed instrument in 96 well format using ForteBio software.
9
Immunostaining of Lung Sections
10
Immunofluorescence Imaging of Oocytes
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Zona pellucida removal was performed by incubating oocytes briefly in acidic MEM-compatible medium (116.4 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 1 mM NaH2PO4, 0.8 mM MgSO4, pH 1.5). Oocytes were then fixed in 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS at room temperature for 30 min, permeabilized in PBS with 0.1% Triton X-100 (Fisher Scientific, BP151–500) for 15 min at room temperature, and blocked in PBS with 0.1% BSA (Sigma-Aldrich, A9647) and 0.01% Tween-20 (Sigma-Aldrich, P7949) for 1 h at room temperature. Anti-Myc primary antibody (Thermo Fisher, MA1–21316) was used at a final concentration of 4 μg/ml. After overnight incubation with anti-Myc antibody, oocytes were washed three times in PBS with 0.1% BSA and 0.1% Tween-20 before incubation with 7.5 μg/ml goat anti-mouse IgG conjugated to Alexa Fluor 488 (Jackson ImmunoResearch) for 1 h at room temperature. Following three washes in PBS with 0.1% BSA and 0.1% Tween-20, oocytes were mounted in VectaShield containing 0.75 μg/ml 4′-6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542). Imaging was performed using a Zeiss Axio Observer Z1 microscope with AxioCam MRm Rev3 camera and ApoTome optical sectioning (Carl Zeiss, Inc.).
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