The crude lysate of U937 cells was prepared by disrupting the cells via sonication (10 cycles) using a picobioruptor (Diagenode) in RIPA buffer (140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 10 mM Tris-Cl pH 8.0, supplemented with 2 X Roche protease inhibitor cocktail, 1 mM PMSF, 2X neutrophil elastase inhibitor (Sigma, M0398). Approximately, 70 μg protein was run in 4–12% Bis-Tris gradient gel (Invitrogen, NP0322BOX), followed by semi-dry transfer to polyvinylidene fluoride (PVDF) membrane (Bio-Rad Transblot). Western blotting was performed using anti-DZIP3 (Sigma, SAB2701600, 1:2,000) and anti-actin (GeneTex, GTX26276, 1:5,000) antibodies. Horse radish peroxidase (HRP)-conjugated anti-rabbit IgG and IR800-conjugated anti-mouse IgG were used as secondary antibodies and the blot was visualized using a Chemidoc system (Bio-Rad).
Np0322box
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NP0322BOX is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a box designed to hold and transport various laboratory items, such as samples, reagents, or instruments. The core function of the NP0322BOX is to provide a secure and protective container for the storage and transportation of laboratory materials.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (6 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (4 mentions)
A22287, by Merck Group (3 mentions)
Gel doc ez system, by Bio-Rad (3 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Rpn2232, by Cytiva (2 mentions)
Imperial protein stain, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Perfection v800 photo color scanner, by Epson (2 mentions)
Mops buffer, by Thermo Fisher Scientific (2 mentions)
M iggk bp hrp, by Santa Cruz Biotechnology (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (2 mentions)
Nitrocelluose membrane, by Cytiva (2 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
β actin, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
42 protocols using np0322box
1
DZIP3 Protein Expression Analysis
2
Actin Polymerization and F-Actin Sedimentation Assays
3
Optimizing Protein Solubility with Detergent and Denaturant Screens
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Bacterial cells were thawed, re-suspended and lysed as before. The crude lysate was incubated with detergents DDM (Sigma Aldrich, D4641), C12E8 (Sigma Aldrich, P8925), Triton-X 100, Tween20 (Sigma Aldrich, P1379), CHAPS (Sigma Aldrich, P9426) and sarkosyl (Sigma Aldrich, L9150) separately at a final concentration of 1% and incubated for up to 4 h at 4 °C with agitation. Lysates were then centrifuged at 20 000 rpm for 40 min at 4 °C. Denaturing treatment was also carried out using 6 M guanidine hydrochloride (GuHCI), 50 mM DTT and separately, 10 M urea, 50 mM DTT under the same conditions as the detergent screen. Pellet and supernatant fractions were run out on an SDS–PAGE protein gel (Invitrogen, NP0322BOX) with simplyblue staining and analysed by Western blot to detect the protein and determine its solubility.
4
Quantifying Cell-Specific Protein Expression
5
Actin Polymerization and F-Actin Sedimentation Assays
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Rabbit skeletal G-actin (Cytoskeleton Inc., AKL99) was resuspended according to manufacturer instructions. Resuspended G-actin were centrifuged at 100,000 × g to remove aggregated monomers. G-actin was then polymerized according to the manufacturer instruction. For low-speed sedimentation assays, F-actin was stabilized with phalloidin (Sigma-Aldrich, A22287), and centrifuged at 20,000 × g to precipitate nonspecific aggregates. F-actin (5 μM) was incubated with increasing concentrations of 6xHis-MBP-MISP (0–5 μM) for 15 min at room temperature. Subsequently, all MISP/F-actin sample series were centrifuged at 10,000 × g for 20 min at room temperature. For high-speed sedimentation assays, MBP-MISP (0.5 μM) was incubated with increasing concentrations of non-stabilized F-actin (0–10 μM) for 2 h at 4°C. Subsequently, all MISP/F-actin sample series were centrifuge at 100,000 × g for 30 min at 4°C. In low- and high-sedimentation assays, the supernatant was carefully removed without disrupting the pellet. Both supernatant and pellet fractions were boiled with samples buffer and run into a 4%–12% NuPAGE gradient gel (Invitrogen, NP0322BOX). Gels were stained with Coomassie blue (Bio-Rad, 1610786) and imaged in a gel imaging system (Bio-Rad, Gel Doc™ EZ System).
6
Affinity Purification of FimA Protein
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Example 11
A 20 μl bed volume of protein A beads (POROS® MabCapture™, Applied Biosystems®, CA, USA) was washed twice in 1 ml PBS by centrifugation (300 g, 1 min) and removal of the supernatant. Beads were coated with antibody by incubating beads with 10 μg antibody in 0.2 ml PBS for 30 minutes and 30 rpm at room temperature. Beads were washed again twice in 1 ml PBS and beads were taken up in 0.4 ml supernatant of a LB overnight culture of A. baumannii. Supernatant was prepared by centrifugation of the bacterial culture at >4000 g for 5 minutes and supernatant was filtered through a 0.2 um filter for syringes (Nalgene #194-2520). The mixture was incubated for 1 h and 30 rpm at room temperature. Beads were washed again twice in 1 ml PBS. Finally, beads were resuspended in 30 μl lysis buffer for NuPAGE® 4%-20% Bis-Tris gels (NP0322BOX, Invitrogen) and incubated at 98° C. for 5 min. The sample was tested for presence of native FimA by immunoblot analysis as described above according to the manufacturer's instructions for denatured, reduced 4%-20% Bis-Tris gels using MES running buffer (IM-8042 Version H, Invitrogen). Rabbit immune serum against FimA was used for detection of FimA.
Results are shown in
7
Western Blot Analysis of IRG1 Protein
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Cells were collected in 600 μL RIPA lysis buffer and stored at −80 °C before protein extraction. Samples were centrifuged at 10 000 g for 10 min at 4 °C and supernatants were harvested. Protein concentrations were measured with Bio‐Rad Protein Assay Dye Reagent Concentrate (500‐0006, Bio‐Rad, Temse, Belgium). Proteins were diluted in RIPA lysis and loading buffers. Heat‐denatured protein samples were separated on 4–12% BisTris‐polyacrylamide gel electrophoresis (NP0322BOX, Invitrogen) followed by transfer to polyvinylidene flouride (PVDF) membranes 0.2 μm (LC2005, Invitrogen). After blocking with 5% (wt/vol) dry milk in TBS containing 0.1% triton (TBST), the membrane was incubated overnight at 4 °C with primary anti‐IRG1 antibody (Ab222411, Abcam) diluted 1 : 250 in 1% (wt/vol) BSA in TBST with constant shaking. After three washing steps with TBST, the membrane was incubated with anti‐rabbit antibody coupled to horseradish peroxidase and revealed by chemoluminescence using Pierce™ ECL detection reagents (Thermo Fisher Scientific). For the second hybridization, the membrane was incubated with anti‐actin antibody (MAB 1501, Millipore, Overijse, Belgium) for 90 min at RT in 1% (wt/vol) BSA in TBST with constant shaking. After three washing steps with TBST, the membrane was incubated with anti‐mouse antibody coupled to horseradish peroxidase and revealed by chemoluminescence.
8
HOXD13 Western Blot Protocol
9
PEG Precipitation for Plasma Proteome Analysis
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PEG precipitation of plasma sample was performed according to the probing protocol described above using 4, 5, and 6 final % of PEG without the addition of competitor or aptamer library. After final re-suspension step sample protein content was measured by nanodrop. Samples (50 μg of recovered plasma sample) were separated on 4–12% Bis Tris protein gel (NP0322BOX, Invitrogen). Control for Ab performance was 0.05 μg exosome sample from the VCaP cell line, isolated by UC. Proteins were transferred to a nitrocellulose membrane (IB301001, Invitrogen) by iBlot standard procedure. Primary antibody probing was performed with 1 μg/mL mouse anti-CD9 ab (MAB1880, R&D Systems) followed by 1:10,000 diluted goat anti-mouse HRP secondary antibody (115-035-062, Jackson Immuno Research) and final development of signal by 5 min incubation with vendor recommended concentration of chemiluminescent substrate (34096, Thermo). Image of blot was captured by imager (PXi, Syngene) following 10 s exposure.
10
Actin Polymerization and MISP Binding Assay
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Rabbit skeletal G-actin (Cytoskeleton Inc., AKL99) was resuspended according to manufacturer instructions. Resuspended G-actin were centrifuged at 100,000 x g to remove aggregated monomers. G-actin was then polymerized according to the manufacturer instruction. F-actin was stabilized with phalloidin (SIGMA, A22287), and centrifuged at 20,000 x g to precipitate nonspecific aggregates. F-actin (5 µM) was incubated with increasing concentrations of 6xHis-MBP-MISP (0-5 µM) for 15 minutes at room temperature. Subsequently, all MISP/F-actin sample series were centrifuged at 10,000 x g for 20 minutes at room temperature. The supernatant was carefully removed without disrupting the pellet. All pellets were boiled with samples buffer and run into a 4-12% NuPAGE gradient gel (Invitrogen, NP0322BOX). Gels were stained with Coomassie blue (Bio-Rad, 1610786) and imaged in a gel imaging system (Bio-Rad, Gel Doc™ EZ System).
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