Ninety-six-well plates were coated with 0.25 μg/ml unlabeled mouse anti-chicken IgA (i.e., total IgA; H+L, ThermoFisher) overnight at 4°C. CON and SPORE SISs were diluted 1:1 in SEA blocking buffer (ThermoFisher), serially diluted 1:2, and incubated for 1 h at RT. Goat-anti-chicken-IgA-AP (H+L, ThermoFisher) was added, followed by PNPP substrate (ThermoFisher), and absorbance was measured at 405 nm. To measure antibody titer, the reciprocal of the highest dilution values doubling the control value (i.e., CON birds) were considered positive. ELISAs were done in duplicate per individual bird (n = 7 per group per time point) and independently-replicated twice.
Sea blocking buffer
Manufactured by Thermo Fisher Scientific
SEA blocking buffer is a laboratory reagent used to prevent non-specific binding in immunoassays. It is designed to effectively block non-specific interactions, thereby improving the specificity and sensitivity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Pnpp substrate, by Thermo Fisher Scientific (2 mentions)
Am tirf mc imaging system, by Leica (2 mentions)
Dylight 488 conjugated donkey α rabbit igg, by BioLegend (2 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
D5628, by Merck Group (1 mentions)
Anti ndufa9, by Abcam (1 mentions)
Mini trans blot cell system, by Bio-Rad (1 mentions)
Immobilon fl 0.45 μm, by Merck Group (1 mentions)
Anti sdha, by Thermo Fisher Scientific (1 mentions)
Anti coi, by Thermo Fisher Scientific (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Pet 101 d topo vectors, by Thermo Fisher Scientific (1 mentions)
8 protocols using sea blocking buffer
1
Quantifying Chicken IgA Antibodies
2
Quantifying NK Cell DAP12 Expression
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Images were taken using Leica AM TIRF MC imaging system as described with the following modifications [108 (link)]. NK-cells were isolated by negative selection and placed on glass chamber slides (5x104 cells/chamber; LabTek II) precoated with 10μg/mL α-Ly49H mAb. Cells were stimulated for 15 minutes, fixed with 4% paraformaldehyde, and permeabilized with 0.25% Triton-X. Cells were blocked with SEA blocking buffer (Thermo-Fisher) for 1 hour and stained with 5 μL rabbit α-human/mouse DAP12 antibody (ab219765, Abcam) overnight at 4°C. Cells were washed and incubated with DyLight 488-conjugated donkey α-rabbit IgG (poly4064, BioLegend) secondary antibody for 2 hrs at room temperature. Cells were washed and fresh PBS was added to each well. Images were taken at room temperature using 100X oil submersion lens and Leica AM TIRF MC imaging system at the University of Iowa Central Microscopy Research Facility. Laser intensity and exposure parameters remained constant within each experiment. TIRF microscopy images were analyzed using ImageJ software. Membrane DAP12 was quantified by measuring mean pixel intensity in the longest axis of cells.
3
Tau protein assessment in neurodegeneration
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Samples were boiled in 1 × LDS loading buffer and 100 mM DTT. A volume corresponding to 0.5 µl S1 and P3 isolated from 24 weeks old rTg4510 and non-transgenic littermate (non-tg) mice or 5 μl S1 and P3 isolated from four pooled Alzheimer’s disease (AD) and healthy control (HC) brains was loaded on a 4–12% Bis-Tris NuPAGE Gel (LifeTech Novex). After electrophoresis, the proteins were blotted over to a Immobilon–FL PVDF membrane (0.45 μm, IPFL10100, Millipore). The membrane was blocked with SEA blocking buffer (Product #37527, Thermo Fisher). Tau and pTau levels were assessed in the samples using 1 μg/mL C5.2 overnight at 4 °C. A secondary fluorophore conjugated IgG antibody was used (IRDye 680 Goat anti-mouse, LICOR biosciences) and the signal was quantified using Odyssey CLx and Image studio software (LI-COR biosciences).
4
ZIKV and Norovirus Biosensor Assay
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The SAM was prepared
using a 1:30 ratio of the protein A/G Cys-tagged recombinant protein
(Prospec pro-1928, concentration of 6.3 μM) and MT(PEG)4 spacer (189 μM, Thermofisher 26132) in ultrapure water.
Following an incubation time of 16 h, the sample was rinsed with PBS.
The mouse anti-Norovirus GI antibody (NativeAntigen MAB12495-100)
at 1 μM and the anti-ZIKV virus antibody mAb ZkE3 at 1 μM
were prepared in PBS and added to separate regions of the sample for
2 h using culture well inserts (Ibidi) to isolate the regions. The
sample was rinsed with PBS, and SEA blocking buffer (Thermo 37527)
was added to the sample for 30 min. The sample was rinsed with PBS,
a fluidic chamber was attached, and measurements were performed. Medium
containing 1.6 × 106 PFU/mL ZIKV was diluted 70:30
(4.8 × 105 PFU/mL) and added to the sample for 1 h.
The sample was rinsed with PBS and measurements were performed. A
Norovirus VLP solution (NativeAntigen REC31722-100) was added to the
sample for 1 h. The sample was rinsed with PBS and measurements were
performed.
using a 1:30 ratio of the protein A/G Cys-tagged recombinant protein
(Prospec pro-1928, concentration of 6.3 μM) and MT(PEG)4 spacer (189 μM, Thermofisher 26132) in ultrapure water.
Following an incubation time of 16 h, the sample was rinsed with PBS.
The mouse anti-Norovirus GI antibody (NativeAntigen MAB12495-100)
at 1 μM and the anti-ZIKV virus antibody mAb ZkE3 at 1 μM
were prepared in PBS and added to separate regions of the sample for
2 h using culture well inserts (Ibidi) to isolate the regions. The
sample was rinsed with PBS, and SEA blocking buffer (Thermo 37527)
was added to the sample for 30 min. The sample was rinsed with PBS,
a fluidic chamber was attached, and measurements were performed. Medium
containing 1.6 × 106 PFU/mL ZIKV was diluted 70:30
(4.8 × 105 PFU/mL) and added to the sample for 1 h.
The sample was rinsed with PBS and measurements were performed. A
Norovirus VLP solution (NativeAntigen REC31722-100) was added to the
sample for 1 h. The sample was rinsed with PBS and measurements were
performed.
5
Quantification of Membrane-Bound AKT in Activated CD8 T Cells
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Images were taken using Leica AM TIRF MC imaging system as described with the following modifications (Bilal et al., 2015 (link)). P14 CD8 T cells were isolated by positive selection, based on Thy1.1, and placed on glass chamber slides (5 × 104 cells/chamber; LabTek II) precoated with 10 μg/mL α-CD3 mAb. Cells were stimulated for 15 minutes, fixed with 4 % paraformaldehyde, and permeabilized with 0.25 % Triton-X. Cells were blocked with SEA blocking buffer (Thermo-Fisher) for 1 hour and stained with 5 µL rabbit α-human/mouse AKT antibody (11E7, Cell Signaling Technology) overnight at 4 °C. Cells were washed and incubated with DyLight 488-conjugated donkey α-rabbit IgG (poly4064, BioLegend) secondary antibody for 2 hr at room temperature. Cells were washed and fresh PBS was added to each well. Images were taken at room temperature using 100 X oil submersion lens and Leica AM TIRF MC imaging system at the University of Iowa Central Microscopy Research Facility. Laser intensity and exposure parameters remained constant within each experiment. TIRF microscopy images were analyzed using ImageJ software. Membrane AKT was quantified by measuring mean pixel intensity in the longest axis of cells.
6
Blue Native Electrophoresis Analysis of Mitochondrial Supercomplexes
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Supercomplex levels and compositions were analyzed in isolated mitochondria from cells by BNE. Mitochondrial proteins were solubilized with 10% digitonin (4 g/g) (D5628, Sigma-Aldrich) and run on a 3 to 13% gradient blue native gel. The gradient gel was prepared in 1.5-mm glass plates using a gradient former connected to a peristaltic pump. Proteins were electroblotted onto polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-FL, 0.45 μm, Merck Millipore, IPFL00010) for 1 hour at 100 V in transfer buffer (48 mM tris, 39 mM glycine, 20% EtOH). A Mini Trans-Blot Cell system (Bio-Rad) was used. Sea Blocking buffer (37527, Thermo Fisher Scientific) or phosphate-buffered saline (PBS) with 5% BSA was used for 1 hour at room temperature (RT) to avoid nonspecific binding of antibodies. For protein detection, antibodies were incubated with the membrane for 2 hours at RT. Secondary antibodies were incubated for 45 min at RT. The membrane was washed with PBS–0.1% Tween 20 for 5 min three times between primary and secondary antibodies, and after secondary antibodies, the last wash was only PBS. To study supercomplex assembly, the PVDF membrane was sequentially probed with complex I (anti-NDUFA9, Abcam), complex IV (anti-COI, Invitrogen), and complex II (anti-SDHA, Thermo Fisher Scientific) antibodies.
7
Immunostaining of hiPSC-derived Cultures
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Confluent hiPSC-derived cultures seeded on glass coverslips were fixed at multiple timespoints with 10% formalin buffered solution for 10 min at room temperature followed by three washes with 1X phosphate-buffered saline (PBS). Cells were permeabilized with 0.2% TritonX-100 in PBS for 15 min, blocked with SEA blocking buffer (37527, Thermo Fisher Scientific) for 1 h, and incubated in primary antibody solution overnight at 4°C. Coverslips were washed three times with 1X PBS and incubated in species appropriate secondary antibodies conjugated to Alexa Fluor 488, 555, or 647 (Thermo Fisher Scientific) at room temperature for 2 h. For nuclei staining, cells were incubated in 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI; 1:1000; Thermo Fisher Scientific) for 5 min and washed two times in 1X PBS. Each staining was performed on three biological replicates per cell line. Staining was performed on all cell lines except AISC-0031-035, which contained an endogenous mRFP tag that interfered with antibody labeling. Negative controls were obtained by omission of the primary antibody. All primary antibodies used are listed in Supplementary Table 2 .
8
Immunogenic Bacterial Factors Elicit IgA Responses
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Ninety-six-well plates were coated with 2.0 μg/ml of lipopolysaccharide (LPS, Salmonella enterica serovar Typhimurium, Sigma), salmochelin receptor (IroN), aerobactin receptor (IutA), or 0.25 μg/ml unlabeled chicken IgA (i.e., total IgA; H+L, Thermo Fisher Scientific) overnight at 4°C. LPS is common in gram-negative bacteria, and IroN and IutA are virulence factors involved in iron acquisition. Recombinant IroN and IutA proteins were purified from culture of E. coli BL21 containing the pET-101/D-TOPO vectors (Invitrogen) carrying iroN or iutA genes as previously described (Mellata et al., 2016 (link)). SISs were diluted 1:1 in SEA blocking buffer (Thermo Fisher Scientific), serially diluted 1:2, and incubated for 1 h at room temperature. Goat-anti-chicken-IgA-AP (H+L, Thermo Fisher Scientific) was added, followed by PNPP substrate (Thermo Fisher Scientific), and absorbance was measured at 405 nm. To measure antibody titer, the reciprocal of the highest dilution values doubling the control value (i.e., CON birds) were considered positive. ELISAs were done in duplicate per individual bird and independently replicated twice.
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