Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed as described previously [99 (link)]. Briefly, VLPs were solubilized in 0.12% Triton X-100 in 1 mM EDTA. An equal volume of 2x sample buffer (100 mM morpholinepropanesulfonic acid (MOPS), 100 mM Tris HCl, pH 7.7, 40% glycerol, and 0.1% Coomassie blue) was added. Samples were then loaded onto a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and separated at 4°C for 3 hours at 100V. Proteins were then transferred to polyvinylidene difluoride (PVDF) membrane, destained, immersed in blocking buffer (4% nonfat milk in PBST) and probed with an anti-gp120 cocktail (39F, F2A3, C011 and 14e at 1μg/ml) and/or an anti-gp41 cocktail (2F5 and 4E10 at 1μg/ml). Blots were then probed by an anti-human Fc alkaline phosphatase conjugate (Accurate Chemicals) and developed using SigmaFast BCIP/NBT substrate (Sigma).
Sigmafast bcip nbt substrate
Manufactured by Merck Group
Sourced in United States
SigmaFast BCIP/NBT substrate is a ready-to-use, chromogenic substrate system for the detection of alkaline phosphatase-conjugated detection systems. It provides a simple, reliable, and consistent method for visualizing target analytes in various immunoassay and histochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (5 mentions)
Anti human fc alkaline phosphatase conjugate, by Jackson ImmunoResearch (2 mentions)
Streptavidin alkaline phosphatase, by Mabtech (2 mentions)
Msips4510, by Merck Group (2 mentions)
Ifnγ elispot pairs, by BD (2 mentions)
Ks elispot system, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ferritin, by GE Healthcare (1 mentions)
Methanol, by Carlo Erba (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Cl5031, by Cedarlane (1 mentions)
C5275, by Merck Group (1 mentions)
Peptide n glycosidase f pngase f, by New England Biolabs (1 mentions)
Nh osteodiff medium, by Miltenyi Biotec (1 mentions)
Nh adipodiff medium, by Miltenyi Biotec (1 mentions)
Oil red o, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Alkaline phosphatase, by Merck Group (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
0.2 m nitrocellulose membrane, by Bio-Rad (1 mentions)
18 protocols using sigmafast bcip nbt substrate
1
BN-PAGE Analysis of HIV-1 VLPs
2
BN-PAGE Assay for Antibody Binding and Depletion of Viral Proteins
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Blue native PAGE (BN-PAGE) was performed as described previously [3 (link),50 (link),51 (link),53 (link)]. Briefly, VLPs were solubilized in 0.12% Triton X-100 in 1 mM EDTA. An equal volume of 2x sample buffer (100 mM morpholinepropanesulfonic acid (MOPS), 100 mM Tris HCl, pH 7.7, 40% glycerol, and 0.1% Coomassie blue) was added. Samples were then loaded onto a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and separated at 4°C for 3 hours at 100V. The gel was then blotted onto polyvinylidene difluoride membrane, destained, immersed in blocking buffer (4% nonfat milk in PBS) and probed with an anti-gp120 cocktail (mAb b12 and 39F at 1μg/ml) and/or a anti-gp41 cocktail (mAb 2F5, 4E10, 7B2, 2.2B at 1μg/ml). Blots were then probed by an anti-human Fc alkaline phosphatase conjugate (Accurate Chemicals) and developed using SigmaFast BCIP/NBT substrate (Sigma).
BN-PAGE “shift” assays were used to measure the ability of antibodies to bind and deplete the unliganded trimer [3 (link),25 (link),50 (link),51 (link),53 (link)]. VLPs were incubated with mAb or serum for 1h at 37°C, then washed with PBS and resolved by BN-PAGE-Western blot, as above.
BN-PAGE “shift” assays were used to measure the ability of antibodies to bind and deplete the unliganded trimer [3 (link),25 (link),50 (link),51 (link),53 (link)]. VLPs were incubated with mAb or serum for 1h at 37°C, then washed with PBS and resolved by BN-PAGE-Western blot, as above.
3
Measuring Antibody Binding to VLP Env
4
Osteoblast Detection Protocol
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Detection of osteoblasts was performed by removing StemMACS OsteoDiff. Media (Miltenyi Biotec, Germany) and washing the cells twice with 300 μl of sterile PBS (in house reagent). Cells were fixed by adding 300 μl of ice cold methanol (Carlo Erba, France) and incubating 5 minutes at −20°C. methanol was aspirated completely, cells were washed twice with deionisated H2O, and 300 μl of SIGMAFAST BCIP/NBT substrate (Sigma-Aldrich, USA) was added to all wells. Plates were incubated 10 minutes at RT. Substrate was aspirated, cells were washed 2x with deionisated H2O, and finally 100 μl of deionisated H2O was added to keep cells moisture. Immediately after staining stained cells were examined under microscope and pictures were taken with camera. Purple stained cells were considered to be positive.
5
ELISPOT Assay for T Cell Cytokine Secretion
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Enzyme-Linked ImmunoSpot (ELISPOT) assays in 96-well plates (S2EM004M99; Fisher Scientific) were performed according the manufacturer’s guidelines. Antibodies used were anti-interleukin 2 (IL-2) (551876; BD Biosciences) and biotinylated anti-IL-2 (551876; BD Biosciences). Assays were performed in serum-free AIM V Medium (12055-091; Life Technologies). In each well, T cells and antigen-presenting cells (with or without antigen or cell lines, as indicated in each figure legend) were incubated at 37°C (5% CO2) overnight. The number of T cells from the β-cell scaffolds and the control scaffolds ranged from 2,000 to 10,000 per well (unless specifically stated in the figure legend). Plates were developed by incubating with 1:1,000 streptavidin-alkaline phosphatase enzyme conjugate (554065; BD Biosciences) for 45 min and developed using SIGMAFAST BCIP/NBT substrate (B5655; Sigma).
6
Evaluating Epitope-Specific T Cell Responses in Vaccinated Mice
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To evaluate epitope-specific T cell responses, splenocytes were collected from vaccinated mice 7 days after their last immunizations. After splenocytes were purified with lympholyte-M (CL5031, Cedarlane), 0.25 million splenocytes were plated in each well of a 96-well ELISPOT plate (Millipore, MSIPS4510), in 200 μl per well. The cells were either left unstimulated as negative control or stimulated with peptides (1 × 10−3 mM Trp2, or 1 × 10−3 mM Td) or ConA (positive control, C5275, Sigma) for 20 hours at 37°C in a humidified CO2 incubator. IFNγ ELISPOT pairs (551881, BD), streptavidin–alkaline phosphatase (3310-10-1000, Mabtech), and Sigmafast BCIP/NBT substrate (B5655, Sigma) were used to detect and develop antigen-specific spots. Plates were enumerated by ZellNet Consulting using a Zeiss KS ELISPOT system.
7
Western Blot Analysis of Recombinant Proteins
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Standard methods were used to detect proteins by Western blotting (30 ). After resolution on a 4–20% Tris-Glycine gradient pre-cast gel (Thermo Scientific, UK) by SDS-PAGE and transfer to a nitrocellulose membrane, blots were blocked in 3% BSA/PBS overnight and incubated for 1 h at room temperature (RT) with antibody. Tag-specific detection of the hexahistidine sequence was achieved by anti-pentahistidine monoclonal antibody (Sigma-Aldrich, UK). Antigen-specific detection was performed either with pooled day 70 sera (n = 5 BALB/c mice) or 4B7, Pfs25-specific monoclonal antibody (31 (link)). Blots were washed with PBS-Tween 20 (PBS/T) for 30 min before incubation with alkaline-phosphatase conjugated goat-anti-mouse antibody for 1 h. Blots were then washed for 30 min with PBS/T, rinsed with deionized water and incubated with SIGMAFAST™ BCIP®/NBT substrate (Sigma-Aldrich, UK) for direct band visualization. Peptide-N-Glycosidase F (PNGase F) treatment (New England Biolabs, UK) was carried out in accordance with manufacturer's instructions.
8
Influenza A Virus Quantification
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Whole lung tissue was harvested in PBS at the indicated times, homogenized, and stored at − 70 °C. Virus titers were measured with a standard plaque assay by infecting MDCK cell monolayers with serial 5-fold dilutions of lung suspension in duplicate. Eighteen to twenty-four hours after infection, monolayers were washed and fixed with 80% acetone in water. Infected cell clusters were detected with a biotin-labeled mouse anti-influenza A monoclonal antibody (Chemicon), followed by staining with streptavidin-AP, and visualized with Sigma Fast BCIP/NBT substrate (Sigma). The number of viral-foci units (VFUs) was counted, and the data were shown as the VFU/lung.
9
Assessing hASCs Differentiation Potential
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hASCs differentiation capability was assessed by investigating osteogenic and adipogenic induction. For osteogenic differentiation, cells were seeded into six-well culture plates and at a density of 5.0 × 103 cells/cm2 and cultivated in NH OsteoDiff medium (Miltenyi Biotec, GmBH) for two weeks. Osteogenic potential was assessed by alkaline phosphatase activity with SIGMA FAST BCIP/NBT substrate (SIGMA-Aldrich, USA).
For adipogenic differentiation, cells were placed into six-well culture plates at a density of 5.0 × 103 cells/cm2 and cultivated in NH AdipoDiff medium (Miltenyi Biotec, GmBH) for three weeks. Adipogenic potential was assessed by Oil Red O (SIGMA-Aldrich, USA) staining.
For adipogenic differentiation, cells were placed into six-well culture plates at a density of 5.0 × 103 cells/cm2 and cultivated in NH AdipoDiff medium (Miltenyi Biotec, GmBH) for three weeks. Adipogenic potential was assessed by Oil Red O (SIGMA-Aldrich, USA) staining.
10
Detection of Recombinant Protein Expression
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HEK293 cells were infected with either 1 × 105 vp hAd5:tgG-RL, hAd5 control or mock infected. Conditioned media containing 20 ug total protein was isolated from all cultures 72 h post infection and subject to electrophoresis in 10% SDS-polyacrylamide gels, and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were probed with either anti-tgG or anti-RL polyclonal serum at 1:1000 or 1:2000, respectively. Immune complexes were further probed with alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) (KPL) at 1:4000. Specific proteins were visualized following the addition of SigmaFAST BCIP/NBT substrate (Sigma-Aldrich).
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