24-mCAF, CAF, CMF and β-SF were available from previous studies.13 (link),14 (link) A549 human adenocarcinoma epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA). Methylthiazol tetrazolium (MTT), triethylammonium bicarbonate (TEAB) buffer, bovine serum albumin (BSA) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO) and culture flasks and plates from Corning (Corning, NY). Gel and buffer preparation reagents, polyvinylidene difluoride (PDVF) membranes, Mini Trans-Blot Electrophoretic Transfer Cell and alkaline phosphatase conjugate substrate kit were obtained from Bio-Rad (Hercules, CA). Antibodies were purchased from Invitrogen (Carlsbad, CA).
Alkaline phosphatase conjugate substrate kit
Manufactured by Bio-Rad
Sourced in United States
The Alkaline phosphatase conjugate substrate kit is a laboratory tool used to detect and quantify the presence of alkaline phosphatase (ALP) in samples. It provides a substrate for the ALP enzyme, which catalyzes a reaction that produces a colored or luminescent product. The kit includes the necessary reagents and components to perform this ALP detection and quantification assay.
Automatically generated - may contain errors
Lab products found in correlation
Mini trans blot cell, by Bio-Rad (2 mentions)
Multiscreen ip plate, by Merck Group (2 mentions)
Goat anti rabbit igg alkaline phosphatase conjugate, by Bio-Rad (2 mentions)
Mahas4510, by Merck Group (2 mentions)
Mini trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mini protean cell system, by Bio-Rad (1 mentions)
Precision plus protein kaleidoscope, by Bio-Rad (1 mentions)
Recombinant human il 15, by Thermo Fisher Scientific (1 mentions)
Il 15, by Merck Group (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Anti ifnγ clone 1 d1k, by Mabtech (1 mentions)
Anti mouse igg alkaline phosphatase, by Merck Group (1 mentions)
Penta his antibody, by Qiagen (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
M per lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
26 protocols using alkaline phosphatase conjugate substrate kit
1
Cytotoxicity Evaluation of Cancer Cell Lines
2
Arabidopsis Thylakoid Membrane Immunoblotting
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Immunoblotting was performed as described in the protocol of BioRad laboratories. Protein samples from thylakoid membranes isolated from leaves of 5 to 6-week-old Arabidopsis plants of WT or α-CA2-KO were separated by electrophoresis in a 16% polyacrylamide gel under denaturing conditions using the Mini-PROTEAN Cell system (BioRad, Hercules, CA, USA). Precision Plus Protein Kaleidoscope (10–250 kDa) (BioRad, Hercules, CA, USA) was used as protein molecular weight markers. After electrophoresis, the proteins were transferred onto a PVDF membrane (BioRad, Hercules, CA, USA) using a wet blotting system Mini Trans-Blot Cell (BioRad, Hercules, CA, USA). Immunoblotting was carried out using primary polyclonal rabbit antibodies against D1 and PsaC proteins (Agrisera, Vännäs, Sweden) and secondary goat anti-rabbit IgG, AP conjugated antibodies (BioRad, Hercules, CA, USA). The membranes were visualized using an alkaline phosphatase conjugate substrate kit (BioRad, Hercules, CA, USA). The PVDF membranes were scanned on a flatbed scanner in transmission mode for further analysis. Quantification of the optical density of the bands on the blots was performed in the ImageJ software. The results of the Western-blot analysis were obtained from two independent experiments.
3
SARS-CoV-2 Antigen-Specific T-Cell Responses
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Multiscreen-IP plates (Millipore-Sigma) were coated overnight at 4°C with 2μg/mL anti-IFN-γ (clone 1-D1K, Mabtech) washed with sterile PBS, and blocked with complete RPMI-10% FBS. PBMC isolated from Neuro-PASC, COVID convalescent, and healthy control subjects were used either freshly isolated or after thawing and resting overnight in media containing 10ng/μL recombinant human IL-15 (Peprotech) at 37°C, 5% CO2. Cells were then plated at a concentration of 2.5x105 cells/well in 200μL of media and stimulated with the indicated antigen mixtures from SARS-CoV-2 at a concentration of 2μg/mL in complete RPMI medium containing 5% human AB serum (Sigma-Aldrich) and 5ng/mL IL-15. Plates were incubated at 37°C, 5% CO2 for 20h and washed 5x with dH2O and PBS-0.05% Tween-20 (PBS-T). 2μg/mL biotinylated IFN-γ (clone 7-B6-1, Mabtech) diluted in PBS-10% FBS (PBS-F) was added to the respective wells and plates were incubated for 1.5h at RT. Plates were subsequently incubated for 40 minutes at RT in streptavidin-alkaline phosphatase in PBS-F (Jackson ImmunoResearch) was added after washing plates 5x in PBS-T. ELISPOT plates were developed using an Alkaline Phosphatase Conjugate Substrate Kit according to manufacturer’s instructions (Bio-Rad Laboratories, Carlsbad, CA). IFN-γ producing cells were quantified using an ImmunoSpot plate reader (Cellular Technologies, Ltd., Shaker Heights, OH).
4
Recombinant ALDH10s Expression in E. coli
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The recombinant Arabidopsis thaliana [L.] Heynh. ALDH10s were induced in transformed Escherichia coli strain BL21 cells (EMD Millipore) by the addition of isopropyl β-D-1-thiogalactopyranoside (final concentration of 0.4 mM) to the Lysogeny broth medium containing 50 μg mL−1 ampicillin when the cell culture had an OD600 of 0.5. The cells were collected 4 h after induction by centrifugation at 5000× g for 5 min and then stored at −80 °C. Bacterial lysis and purification of the recombinant protein by affinity chromatography were conducted as described previously7 (link). High-protein fractions were combined and precipitated on ice by slowly adding solid ammonium sulfate, with gentle stirring, to 80% saturation. Aliquots of precipitated protein were stored at −80 °C. Total proteins were separated by SDS-PAGE gel electrophoresis and visiualized by staining with Coomassie Blue R-250 using standard protocols33 (link). Immunoblot analysis was based on a semi-dry method using a mouse monoclonal IgG against the His tag (Santa Cruz Biotechnology, 1:1000) and an anti-mouse IgG–Alkaline Phosphatase (Sigma 1:10000) as primary and secondary antibodies, respectively. Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit was used to detect fusion proteins.
5
Western Blot Protein Detection Protocol
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Following SDS-PAGE, proteins were transferred onto PVDF membrane and blocked in 5 % skim milk (1X Tris buffered saline, TBS) at room temperature for 1 h. Primary antibodies were prepared at 1:5000 dilutions of Penta His antibody (Qiagen Cat No:- 34460), 1:2000 dilution of Anti-Pfs25 mAb 4B7 (BEI), or 1:1000 dilutions of 1G2 (final 2 µg/ml) in 1 % skim milk in 1X TBS buffer containing 0.05 % Tween-20 (TBS-T). Blots were incubated in the primary antibody solution for 1 h at room temperature or overnight at 2–8 °C. Membranes were washed with 1X TBS-T buffer (3X for 10 min) and secondary antibody 1:1000 dilution of goat anti mouse IgG-HRP (Santa Cruz) or 1:4000 goat anti mouse IgG Alkaline Phosphatase (BioRad) in 1 % skim milk (1X TBS-T buffer) was added and incubated at room temperature for 1 h. Membranes were then again washed with 1X TBS-T buffer (3X for 10 min). HRP labeled blots were developed with TMB (3,3,5,5′-Tetramethylbenzidine) substrate. Alkaline Phosphatase labeled blots were developed using Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit.
6
Western Blot Analysis of Somatostatin Receptors
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For western blot analysis adherent cells were harvested in M-PER lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor (Sigma-Aldrich, St. Louis, IL, USA). Total protein concentration was measured by the Pierce BCA protein assay kit (Thermo Fisher Scientific). SDS-PAGE gel electrophoresis was performed under reducing conditions using 10% polyacrylamide gels. Equal amounts of proteins (40 μg) were separated and then transferred to PVDF membrane using wet transfer. After blocking with 5% TBST-milk, the membranes were incubated (overnight, 4 °C) with primary antibodies SSTR-5: anti-somatostatin-receptor-2-rabbit, 1:1000 dilution (ab134152 rabbit monoclonal; Abcam, Cambridge, UK) and anti-somatostatin-receptor-5-rabbit, 1:1000 dilution (ab109495 rabbit monoclonal; Abcam). As a housekeeping gene anti-HPRT was used, 1:1000 dilution (PA-22281 rabbit polyclonal; Cell Signaling Technology, Danvers, MA, USA). After the washing steps, the membrane was incubated with alkaline phosphatase conjugated polyclonal rabbit-anti-mouse secondary antibody, 1:3000 dilution (sc-2771; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Proteins were detected with Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad).
7
Protein Expression Profiling in HUVECs
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HUVECs were washed with PBS and lysed with mammalian tissue lysis/extraction reagent including protease inhibitor (Roche, IN, USA). Protein concentrations were determined using BCA protein assay kit (Pierce Chemicals, CA, USA), and 1× sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% bromophenol blue) was added. Proteins were separated by SDS-polyacrylamide gel electrophoresis in 10–15% gels, transferred onto polyvinylidene difluoride membranes, and incubated with anti-p16 (ab54210, Abcam, MA, USA), anti-ATF4 (11815, Cell Signaling, MA, USA), anti-peIF2α (9721, Cell Signaling, MA, USA), anti-NOX4 (LS-C313066, Lifespan Biosciences, WA, USA), anti-NLRP3 (ab214185, Abcam, MA, USA), anti-cleaved caspase-1 (4199, Cell signaling, MA, USA), anti-GAPDH (2118, Cell signaling, MA, USA), and anti-tubulin (3873, Cell signaling, MA, USA), antibodies at 4 °C overnight. Alkaline phosphatase-conjugated goat anti-rabbit (A120-101AP, Bethyl, TX, USA) or anti-mouse secondary antibody (A90-116AP, Bethyl, TX, USA) was applied for 1 hour at room temperature, and membranes were developed using an alkaline phosphatase conjugate substrate kit (Bio-Rad, CA, USA). Developed protein bands were quantified using Image J (NIH, Bethesda, MD, USA).
8
Western Blot Analysis of Bacterial Proteins
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Proteins extracted from the bacterial stroma and growth medium after fractionation as previously described were separated on a 12% w/v polyacrylamide SDS-PAGE gel. After separation, proteins were transferred onto nitrocellulose membranes at 50 mA for 1h in Tris base (0.192 M) transfer buffer containing glycine (14.5% g/L w/v) and methanol (20% v/v) using a Bio-Rad Mini TransBlot Electrophoretic Transfer Cell system. Membranes were then air-dried and immersed for 2 h in blocking buffer (Tris buffer saline (TBS): 5 × 10−5 M, 0.15 M NaCl, 5% whole milk). Membranes were incubated with primary antibody raised against bacterial EfTu6 (1:100 in blocking buffer) at room temperature for 2 h while shaking. After incubation, the blot was washed three times in 1x TBS supplemented with 0.05% (w/v) Tween 20 for 30 min and incubated for 1.5 h with shaking with secondary antibody diluted 1/5000 (goat anti-rabbit IgG alkaline phosphatase conjugate, Bio-Rad, Schiltigheim, France).). Electrophoretic bands were detected using an alkaline phosphatase conjugate substrate kit (Bio-Rad, Schiltigheim, France).
9
Denaturing Electrophoresis and Western Blot Analysis
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Denaturing electrophoresis was performed according to Schägger and von Jagow (1987) (link) in 15% polyacrylamide gel (PAAG) in Mini-PROTEAN Cell (BioRad). Samples of thylakoid membranes were diluted in the loading buffer (pH 6.8), containing 60 mM of Tris-HCl, 2% sodium dodecyl sulfate, 10% sucrose, 0.05% bromophenol blue, and 5% dithiothreitol, heated at 99°C for 2 min. Insolubilized material was precipitated by centrifugation at 10,000 rpm in Centrifuge MiniSpin (Eppendorf) for 10 min. Samples of denatured proteins from Thyl and STM corresponding to 3 μg of Chl content were loaded on gel. Prestained standard kit in dual color (10–250 kDa) (Bio-Rad, United States) was used as the protein molecular mass markers.
After electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (BioRad, United States) using wet blotting system Mini Trans-Blot Cell (BioRad, United States). Western blot analysis was performed according to Onda et al. (2000) (link) with anti-rabbit primary antibodies against PsbA and PsaC (Agrisera) (AS05 084 and AS10 939, correspondingly). Goat anti-rabbit antibodies labeled with alkaline phosphatase (Agrisera) were used as secondary antibodies in dilution of 1:5,000. The antibody–antigen conjugates were detected by Alkaline Phosphatase Conjugate Substrate Kit (BioRad, United States).
After electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (BioRad, United States) using wet blotting system Mini Trans-Blot Cell (BioRad, United States). Western blot analysis was performed according to Onda et al. (2000) (link) with anti-rabbit primary antibodies against PsbA and PsaC (Agrisera) (AS05 084 and AS10 939, correspondingly). Goat anti-rabbit antibodies labeled with alkaline phosphatase (Agrisera) were used as secondary antibodies in dilution of 1:5,000. The antibody–antigen conjugates were detected by Alkaline Phosphatase Conjugate Substrate Kit (BioRad, United States).
10
Glucagon Detection and Quantification
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Purified GST-FLAG-Glucagon and non-purified protein concentrations were estimated using the Bradford method (1976 (link)). Protein molecular mass was estimated as previously described. Glucagon was detected using the polyclonal anti-glucagon antibody (Santa Cruz, CA., USA) and alkaline phosphatase-conjugated anti-goat IgG (GE Healthcare Inc., Sweden). The Alkaline Phosphatase Conjugate Substrate Kit (BioRad, CA., USA) was used according to the manufacturer’s instructions.
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