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12 protocols using pierce protein free pbs blocking buffer

1

Antibody Binding Assay for Malaria Antigens

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Antibody binding to PcCSP or PcTRAP in macaque plasma were measured by an IgG enzyme linked immunosorbent assay (ELISA) using previously described methods [18 (link),19 (link),20 (link)]. Briefly, Nunc Maxisorp Immuno ELISA plates coated with PcCSP or PcTRAP antigens diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature (RT) overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with PierceTM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, U.S.) for 2 h at RT. Macaque plasma was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected following a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for monkey IgG (Sigma Aldrich, SLM, U.S.). Following additional 6 washes with PBS/T, development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and c using a CLARIOstar instrument (BMG Labtech, Aylesbury, GB). Log reciprocal antibody titers were defined by an absorbance value three standard deviations greater than the average OD405 of the control. The antibody titers were analyzed by unpaired t-test to determine the p-values.
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2

Quantifying Antibody Response to CHIKV E2

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Antibody binding to CHIKV E2 was measured by an IgG enzyme linked immunosorbent assay (ELISA). Briefly, mice sera were diluted in Nunc Maxisorp Immuno ELISA plates coated with E2 diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with PierceTM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at RT. Mice serum was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected following a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for whole mouse IgG (A3562-5ML, Sigma Aldrich, St. Louis, MO, USA). Following additional 6 washes with PBS/T, development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and analysed using a CLARIOstar instrument (BMG Labtech, Aylesbury, GB). Serum antibody endpoint titres were defined by an absorbance value three standard deviations greater than the average OD405 of the control.
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3

MAYV E2 and CHIKV E2 Antibodies ELISA

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Specific antibody binding to MAYV E2 or CHIKV E2 was measured by an IgG enzyme linked immunosorbent assay (ELISA) as previously described [24 (link),25 (link)]. Briefly, mice sera were diluted in Nunc Maxisorp Immuno ELISA plates coated with the MAYV E2 or CHIKV E2 diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature (RT) overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with PierceTM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at RT. Mouse serum was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected after a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for whole mouse IgG (A3562-5ML, Sigma Aldrich, St. Louis, MO, USA). Development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and analysed using a CLARIOstar instrument (BMG Labtech, Aylesbury, UK). Serum antibody endpoint titers were defined by an absorbance value three standard deviations greater than the average OD405 of the control.
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4

Western Blot Analysis of AEBP1 Protein Levels

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Cells were lysed in 350 uL RIPA buffer and lysate was transferred to 1.5 ml tubes and centrifuged at 5000 x g for 5 minutes at 4°C. Total protein concentrations for each sample were determined using the Pierce BCA Protein Assay kit (ThermoFisher Scientific). Levels of AEBP1 and GAPDH were detected using western blot analysis. An equal amount of protein was loaded on a NuPage pre-cast Bis-Tris protein gel with a 4–12% polyacrylamide gradient (Life Technologies), and then transferred from gels to nitrocellulose membranes (Life Technologies). Membranes were placed in Pierce Protein-Free (PBS) Blocking Buffer (ThermoFisher Scientific) for ninety minutes at room temperature, and then incubated overnight at 4°C with primary antibodies directed against ACLP/AEBP1 (anti-mouse 1:1000 dilution; Santa Cruz; Dallas, TX; catalog number: sc-271374) or GAPDH (anti-rabbit 1:1000; Cell Signaling Technology; Danvers, MA). Membranes were washed for eight minutes in 1X TBS-Tween buffer, and then incubated with secondary antibodies, either anti-mouse or anti-rabbit, labeled with horseradish peroxidase (1:3000; Cell Signaling Technology) for one hour at room temperature. Membranes were incubated with Clarity Western ECL Blotting Substrate (Bio-Rad; Hercules, CA) and protein bands were imaged using the Odyssey FC imaging system (LI-COR Biotechnology; Lincoln, NE).
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5

Determining ErbB2 Aptamer Binding Affinity

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The equilibrium dissociation constant (Kd) values of ErbB2 aptamer were determined using an enzyme-linked oligonucleotide assay (ELONA). Briefly, 96-well ELISA plates (Corning®, Sigma-Aldrich) were coated with ErbB2 (Cat. No. 1129-ER, R&D system) at 4 ºC for 16 h. The treated wells were blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, followed by three washes with PBS. The biotin-labeled aptamers were added and incubated for 1 h at room temperature. After extensive washing with washing buffer (10 mM PBS, 0.05% Tween-20, pH 7.4), Pierce High Sensitivity Streptavidin HRP (Thermo Fisher Scientific) was added to each well to bind biotin-conjugated aptamer. After incubation for 1 h at room temperature, the plates were washed again as described above. Tetramethyl benzidine (TMB) substrate solution (Thermo Fisher Scientific) was added to each well and incubated for 30 min at room temperature. Then, the reaction was quenched by addition of 2 M H2SO4. The protein-bound aptamer-streptavidin complexes were quantified by determining the absorbance at 450 nm using GloMax® Discover System (Promega, Madison, WI, USA). The saturation curve was plotted and Kd was analyzed with Sigma Plot 12.5 (https://systatsoftware.com/products/sigmaplot/) software.
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6

Quantifying Anti-MuSK Antibodies in Mice

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Serum samples in NSG mice with Nalm-6 13-3B5* were collected at day 5 and day 15 after target cell injection in K2EDTA tubes for MuSK antibody ELISA. To detect human anti-MuSK IgG4, the histidine-tagged recombinant extracellular domain of human MuSK (aa 24–495, R&D Systems, catalog no. 10189-MK) was coated on ELISA plates in PBS overnight at 4 °C at a concentration of 5 µg ml−1. Plates were washed with washing buffer (Invitrogen, catalog no. 00-0400-59), and blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo Scientific, catalog no. 37572). Mouse serum samples were evaluated at a dilution of 1:50 to 1:100 in comparison to a 13-3B5 purified recombinant human monoclonal IgG4 antibody as a reference standard for quantitation. Antihuman IgG (H+L) HRP (Bethyl, catalog no. A80-119P) was used to detect human antibodies. Plates were protected from the light and placed in the dark for 2 h. After washing plates three times, 100 μl of TMB (Thermo Scientific, 34028) was added for 30 min. Plate reading were conducted using ELISA reader (Tecan, Infinite F-50) within 15 min after adding stop solution (Invitrogen, SS04).
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7

Quantifying Anti-MuSK Antibodies in Mice

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Serum samples in NSG mice with Nalm-6 13-3B5* were collected at day 5 and day 15 after target cell injection in K2EDTA tubes for MuSK antibody ELISA. To detect human anti-MuSK IgG4, the histidine-tagged recombinant extracellular domain of human MuSK (aa 24–495, R&D Systems, catalog no. 10189-MK) was coated on ELISA plates in PBS overnight at 4 °C at a concentration of 5 μg ml−1. Plates were washed with washing buffer (Invitrogen, catalog no. 00-0400-59), and blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo Scientific, catalog no. 37572). Mouse serum samples were evaluated at a dilution of 1:50 to 1:100 in comparison to a 13-3B5 purified recombinant human monoclonal IgG4 antibody as a reference standard for quantitation. Antihuman IgG (H+L) HRP (Bethyl, catalog no. A80-119P) was used to detect human antibodies. Plates were protected from the light and placed in the dark for 2 h. After washing plates three times, 100 μl of TMB (Thermo Scientific, 34028) was added for 30 min. Plate reading were conducted using ELISA reader (Tecan, Infinite F-50) within 15 min after adding stop solution (Invitrogen, SS04).
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8

Measuring Neutralizing Antibodies Against AAV

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Female 7–8 weeks old C57BL/6 mice (n = 4 per group) were intravenously administered with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐eGFP or RBC‐AAV‐eGFP. Four weeks after the initial AAV exposure, the same mice were intravenously injected with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Blood was collected on days 0, 27, 42, and 56 post the first injection to quantify the amount of nAbs. nAb titer was measured by ELISA. In brief, 96‐well plate was coated with AAV‐eGFP (2.5 × 109 vg/well in 100 µL PBS) overnight at 4 °C. Plates were then washed 6 times with washing buffer (PBS + 0.01% Tween‐20) and blocked using 300 µL of Pierce Protein‐Free (PBS) Blocking Buffer (Thermo Scientific) for 2 h. After washing the plates 6 times, 100 µL of serum at a serial dilution was added to each well and incubated for 2 h at room temperature. The plates were then washed again and goat anti‐mouse IgG HRP in 100 µL of PBS (1:10 000 dilution) was added to each well for incubation for 1 h. After washing the plates, 100 µL TMB substrate was added to each well; 10 min later, the reaction was stopped by adding 100 µL stopping buffer. Absorbance at 450 nm was measured using a plate reader. The antibody titer (EC50) was defined as the dilution factor at which the absorbance value dropped to half of the maximum.
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9

Lipid Binding and ELISA Assays for Influenza

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Prior to all incubations, 1 μL of purified HKx31 virus and the recombinant H3 protein of HKx31 (Sino Biological) were spotted on the lipid strips as the binding controls. Either PIP or Membrane lipid strip (Echelon Biosciences) was blocked with 50% Pierce Protein-Free (PBS) Blocking Buffer (ThermoFisher) in PBS for one hour and incubated with 1 μg/mL of each monoclonal antibody diluted in the blocking buffer. After a one-hour incubation, the strips were washed three times with PBST and hybridized with the corresponding secondary antibodies as used in Western blot in the protein-free blocking buffer for one hour. The detection of the binding antibodies was described previously in Western blot.
For the PI(4)P ELISA, MaxiSorp 96-well ELISA plates (Invitrogen #44–2404-21) were coated with PI(4)P (Avanti #840045) in absolute ethanol and dried overnight at room temperature. The plates were blocked with 50% Pierce Protein-Free (PBS) Blocking Buffer in PBS for one hour. Serum samples were diluted in PBST with 2.5% FBS, and 75 μL of diluted sample was incubated on the blocked plates for one hour at room temperature. The plate washing and substrate development were as described in the ELISA protocol for influenza antibody detection above.
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10

Quantifying SP-555 Binding to ACE2

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ACE2 (recombinant mouse (HEK293cells; cat#230-30172) and recombinant human ACE2 (HEC 293 cells; cat# 230-30165), RayBiotechLife, Inc., Peachtree Corners, GA), dissolved in carbonate/bicarbonate buffer, was immobilized (25 μg ml−1) on glass slides for 1 h at room temperature (RT), blocked with a non-protein buffer (Pierce Protein-free (PBS) blocking buffer, Cat# 37572, ThermoFisher Scientific), washed, incubated with SP-555 at different concentrations (0.01–1 μg ml−1) in Hank’s buffered salt solution (HBSS) for 1 h at RT, washed, mounted, and imaged. SP-555 intensity from ten fields in two slides for each concentration were analyzed and expressed as intensity per μm2.
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