Protein was extracted from cells growing on 6-well plates with or without treatment with BT#9. Cell and tissue lysates were prepared in the lysis buffer (50 mm Tris-HCl, pH 8.0, 0.1% SDS, 150 mm NaCl, 1% Nonidet P-40, protease inhibitor mixture: 1 μg/mL aprotinin, 1 μg of leupeptin, and 1.0 mm PMSF). Equal amounts of protein were loaded to 10% SDS-PAGE and transferred to a nitrocellulose membrane by tank blotting system (Bio-Rad, Hercules, CA, USA). Membranes were incubated with anti-Magmas antibody overnight followed by HRP-conjugated secondary antibodies. The expression of the protein, β-actin, was used as a loading control. The polyclonal anti-Magmas antibody was a kind gift from Paul T. Jubinsky, M.D., Ph.D. and Mary Short, Ph.D., Albert Einstein Medical College, New York. The Caspase-3, Anti-β-actin, the Anti-rabbit IgG, HRP-linked and Anti-mouse IgG, HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); catalog numbers 9669, 3700, 7074 and 7076, respectively. Thermo Scientific 1-Step Ultra TMB-Blotting Solution (catalog number # 37574) was used to determine the protein band intensity.
1 step ultra tmb blotting solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
1-Step Ultra TMB-Blotting Solution is a ready-to-use substrate solution for the detection of horseradish peroxidase (HRP) in Western blotting applications. It is a stable, single-component solution that produces a blue color upon reaction with HRP.
Automatically generated - may contain errors
Lab products found in correlation
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (3 mentions)
Maxisorp microplate, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Pierce coomassie bradford protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
T9039 10pak, by Thermo Fisher Scientific (2 mentions)
Mcherry antibody, by Thermo Fisher Scientific (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tank blotting system, by Bio-Rad (1 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Goat anti mouse hrp secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pierce protein concentrator columns, by Thermo Fisher Scientific (1 mentions)
Ab97057, by Abcam (1 mentions)
27 protocols using 1 step ultra tmb blotting solution
1
Magmas Protein Expression Analysis
2
SARS-CoV-2 Antibody ELISA Detection
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Ninety-six-well Maxisorp microplates (Nunc) were incubated with 2 μg/ml of recombinant RBD [5 (link),32 (link)] or ectodomain of the S protein, the N protein or with PBS at 4 °C overnight and were then incubated with 5% skim milk in PBS containing 0.05% tween-20 (PBS-T) for 1 h at room temperature. Serum or plasma samples were initially diluted 40-fold in PBS-T containing 5% skim milk, and subsequently serially 4-fold diluted (40- to 2,621,440-fold). The microplates were reacted for 1 h at room temperature with the diluted serum or plasma samples in duplicate, followed by peroxidase-conjugated goat anti-human IgG, Fcγ Fragment specific antibody (Jackson Immuno-Research) or anti-human IgM, Fcμ Fragment specific antibody (Jackson Immuno-Research). 1-Step Ultra TMB-Blotting Solution (Thermo fisher scientific) was then added to each well and incubated for 3 min at room temperature. The reaction was stopped by the addition of 2 M H2SO4 and the optical density at 450 nm (OD450) was immediately measured. The average OD450 values of two PBS-wells were subtracted from the average OD450 values of two RBD-, ectodomain-, or N protein-wells for background correction. A subtracted OD450 value of 0.1 or more was regarded as positive, and the maximum dilution to give a positive result was used as the ELISA titer.
3
Western Blot Analysis of Hla Protein
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Bacterial broth was concentrated 10x in PBS on Pierce Protein Concentrator columns (3K MWCO, Thermo Fisher Scientific, Waltham, USA) and run on 4–12% SDS-PAGE gels. Gels were either stained with Coomassie Brilliant Blue or blotted to nitrocellulose filters for western blot. The western blot was blocked with 2% milk in TBS+0.05% Tween and detected with a mouse anti-Hla primary antibody (8B7, ab190467; Abcam, Cambridge, UK) diluted 1:1000 and a goat anti-mouse-HRP secondary antibody (A28177; Thermo Fisher Scientific) diluted 1:2000, both in TBS+0.05% Tween. Bands were visualized using the 1-Step Ultra TMB-Blotting Solution (Thermo Fisher Scientific). The blot was scanned and bands quantified in Photoshop using integrated density measurements.
4
Immunoblotting Analysis of LRP6 and Actin
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Cells were lysed in 0.5 mL of Laemmli buffer (2×) (Sigma-Aldrich) that contained 200 mM dithiothreitol (DTT, Thermo Fisher Scientific). The cell lysates were then homogenized via sonication for 15 s (Branson 450 Probe Sonifier), boiled at 95°C for 10 min, centrifuged at 16,000 × g for 2 min, and loaded onto Novex WedgeWell 4 to 20% Tris-Glycine gels (Invitrogen). Western blot analyses were performed using either rabbit monoclonal anti-LRP6 (C5C7, rabbit MAb, cell signaling, 1:100, overnight), mouse monoclonal anti-actin (A2066, Sigma-Aldrich, 1:3000, overnight), or anti-HA high-affinity rat MAb (3F10, Roche; 1:2000, overnight). A horseradish peroxidase (HRP)-conjugated goat anti-rabbit (P0448, Dako, 1:3000, 2 h), goat anti-mouse (P0447, Dako, 1:3000, 2 h), or goat anti-rat (Abcam; ab97057; 1:3000, 2 h) was used as the secondary antibody. Signals were detected on nitrocellulose membranes (Thermo Fisher Scientific) using a chemical colorimetric substrate (1-Step Ultra TMB-Blotting Solution, Thermo Fisher Scientific), following the manufacturer’s instructions. Pictures were taken with a Quantum imaging system (Vilber Lourmat) and quantified using the built-in Bio-1D analysis software.
5
Characterization of Burkholderia LPS Profiles
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Purified LPS (10 μg each) from wild-type strains 1026b and 576a and an equal amount of heat-killed lysate from Bp82 and 576mn were separated on SDS-polyacrylamide gels with a 12% resolving gel and a 4% stacking gel. Silver stains were carried out with the Pierce Silver Stain Kit (Thermo Scientific). Coomassie stains were performed using established methods. Colorimetric Western blots were performed by semi-dry electroblotting of SDS-PAGE run gels onto methanol soaked Immobilon PSQ PVDF membranes from Millipore™ or Odyssey nitrocellulose membranes from LI-COR™. Blots were washed with 1xPBS, blocked with 1% skim milk in PBS and detected with 1-Step Ultra TMB-Blotting Solution (Thermo Scientific™) following standard practices and manufacturer’s instructions. Type A LPS mAb 4C7-HRP and type B mAb 5B4-HRP were kindly provided by Dr. David AuCoin and as previously described [24 ]. Rhesus macaque serum from a 1026b aerosol challenged monkey at 28 days post challenge was provided by Battelle, OH and was used at a 1:1000 dilution. Macaque antibodies were detected with anti-monkey IgA, IgG, IgM (H + L)-HRP (Sigma) secondary.
6
Western Blot Analysis of JAK-STAT Pathway
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Cells were seeded (2 × 105) in a six well plate for 24 h and were treated with GE at 150 and 200 (µg/mL) for 24 h followed by washing with pre-cooled PBS. Ice cold RIPA buffer with protease/phosphatase was added for the cell lysis. The lysates were collected and centrifuged at 13,000× g for 30 min at 4 °C. The total protein concentration in cell lysates was measured by the Bradford method. The cell extracts were separated by SDS polyacrylamide gels (8–10%), and then, the protein was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk, incubated with indicated primary and appropriate secondary antibodies (anti-rabbit IgG-HRP conjugates), and protein bands were detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific). STAT1 (D1K9Y) Rabbit mAb, Phospho-STAT1 (Tyr701) (58D6) Rabbit mAb, STAT2 (D9J7L) Rabbit mAb, Phospho-STAT2 (Tyr690) (D3P2P) Rabbit mAb, JAK1 (6G4) Rabbit mAb, Phospho-JAK1(Tyr1034/1035) (D7N4Z) Rabbit mAb, TYK2 (D4I5T) Rabbit mAb, Phospho-TYK2 (Tyr1054/1055) (D7T8A) Rabbit mAb, β-Actin (13E5) Rabbit mAb and Anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Densitometric analysis was performed by Image J software version 1.44.
7
Rapid SARS-CoV-2 Antigen Detection Assay
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Briefly, 10 µl of sample was mixed with 90 µl of running buffer (PBS 1×; 0.5% BSA; 1% Tween®-20; (Sigma, St. Louis, MO, USA) pH = 7.4) and one to five µl of biotinylated, mAb-coated magnetic nanoparticles in a well of a low-binding 96-well plate (Corning, Corning, NY, USA) before being flowed vertically onto a LFIA strip. After 25 min, if brown signals are observed at both test line and control line, a positive result could be concluded. If the signal is only observed at the control line, a signal amplification step is carried out by dipping the test strip into a well containing 30 µl of Streptavidin-PolyHRP80 conjugate at the concentration of 1–10 ng ml−1 (Fitzgerald, Acton, MA, USA). The excess of Streptavidin-PolyHRP80 conjugate was washed by dipping the test strip into a new well containing 50 µl of the running buffer, followed by absorbent pad removal and application of 200 µl of 1-Step™ Ultra TMB-Blotting Solution (Thermo Fisher Scientific, Waltham, MA, USA) on the test strip. The enzyme-substrate reaction was maintained for 10 min at room temperature before reading the results.
8
Agrobacterium-Mediated Protein Extraction
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The agrobacterium-infiltrated leaf tissues were collected using a No. 7 cork borer, flash frozen with N2 liquid, and then ground to fine powders using a FastPrep-24 homogenizer (MP Biomedicals). The ground samples were extracted with 2x Laemmli buffer, boiled for 5 min and subsequently centrifuged at 13,000 rpm for 5 min. The supernatant was loaded onto 12% SDS-PAGE and transferred onto a polyvinylidenedifluoride (PVDF) membrane (Thermo Scientific). The His-tagged Ppal15kDa proteins were detected using HRP conjugated anti-His monoclonal antibody His-probe (H-3) (sc-8036 HRP, Santa Cruz Biotechnology, INC) and 1-Step Ultra TMB-Blotting Solution (Thermo Scientific).
9
Evaluating Recombinant SARS-CoV-2 Protein Purity
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Expression and purity of recombinant S1 and RBD fusion proteins were evaluated by western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified samples were added to sample loading dye NuPAGE LDS sample buffer and reducing agent (both Invitrogen) and heated to 70°C for 10 min. Samples were loaded into pre-cast polyacrylamide gels (Bolt 4–12% Bis-Tris Plus; Invitrogen) and run at 200 V for 40 min. Visualization of protein samples on acrylamide gels was performed using Coomassie Brilliant Blue G250 stain (Merck). Gels were stained overnight with agitation, and destaining solution (30% methanol and 10% acetic acid) was added for 1 h at room temperature. After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane using a dry transblotter (Invitrogen). The membrane was blocked for 30 min (PBS containing 5% fat free milk and 0.1% Tween 20) at room temperature, followed by incubation with mouse anti-rabbit horseradish peroxidase (1:5,000; Sigma) for 1 h at 37°C with agitation. The membrane was washed four times using wash buffer (PBS with 0.1% Tween 20) at room temperature for 15 min, and developed using TMB solution (1-step ultra TMB-blotting solution, Thermo Scientific) in the dark for 30 min.
10
Western Blot Detection of Rus Protein
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Membranes were blocked by incubation in TBS-T (19.3 mM Tris, 150 mM NaCl, 0.1% [v/v] Tween 20, pH 7.6) with 5% (w/v) non-fat dried milk powder for 60 min under gentle shaking. The primary rabbit anti-Rus antibody was diluted 1:1000 in TBS-T containing 5% (w/v) non-fat dried milk powder. Rabbit anti-Rus antibodies were kindly provided by Marianne Ilbert (Bioenergetic and Protein Engineering Laboratory, BIP, Institute of Microbiology of the Mediterranean, IMM). Membranes were incubated with primary antibodies for 1 h under gentle shaking. Afterwards, membranes were washed for a total of 30 min with TBS-T and incubated with Pierce goat anti-rabbit IgG-HRP secondary antibodies (Thermo Scientific) for 1 h under gentle shaking. Secondary antibodies were diluted 1:5000 with TBS-T containing 5% (w/v) non-fat dried milk powder. After a second washing step with TBS-T, 1-Step Ultra TMB-Blotting Solution (Thermo Scientific) was added for colorimetric detection. Membranes were visualized with a ChemiDoc XRS + gel imaging system (Bio-Rad) running ImageLab software (Bio-Rad). Unprocessed images are available in Supplementary Figs. 9 and 11 .
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