ELISA was conducted utilizing lysates from 4-month-old optic nerves, following previously established procedures [40 (link)]. In brief, the optic nerves were homogenized in 300 μL of complete extraction buffer (Abcam, Waltham, MA, USA, ab193970). Subsequently, the ELISA was performed in 96-well microtiter plates (Sigma-Aldrich, Burlington, MA, USA, M9410-1CS) coated with tissue lysates and then left to incubate overnight at 4 °C. The plates were subsequently blocked with 5% BSA for 2 h. After PBS washing, 50 μL of IL1β antibody (Abcam, Waltham, MA, USA, ab9722) at a 1:1000 dilution was added to each well and allowed to incubate for 2 h at room temperature. The cytokines bound to the plate were detected using secondary IgG-HRP (KPL, Sera Care, Milford, MA, USA, 074–1506). TMB substrate solution (Thermo Fisher Scientific, Waltham, MA, USA, 34021) was used to develop color, and the reaction was stopped with 2 N H2SO4 solution (Thermo Fisher Scientific, Waltham, MA, USA, MK-H381-1). The absorbance was measured at 450 nm using a microplate reader [40 (link)].
2 n h2so4 solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
2 N H2SO4 solution is a standardized sulfuric acid solution with a concentration of 2 normal (2 N). This solution is commonly used as a chemical reagent in various laboratory applications, such as pH adjustment, titrations, and other analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Tmb substrate solution, by Thermo Fisher Scientific (2 mentions)
Il 1β antibody, by Abcam (1 mentions)
Ab193970, by Abcam (1 mentions)
Human progranulin duoset elisa, by R&D Systems (1 mentions)
Hrp conjugated goat anti mouse igg h l, by Southern Biotech (1 mentions)
1 step ultra tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Synergy h4 microplate reader, by Agilent Technologies (1 mentions)
3 protocols using 2 n h2so4 solution
1
Optic Nerve ELISA for IL1β Quantification
2
PGRN Levels Quantification by ELISA
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To determine the human PGRN levels in the serum, frontal cortex, and spinal cord lysates, homemade rabbit anti-human granulin E antibodies were used to coat a 96-well microplate with 100 μL per well and incubate overnight at 4°C. The next day, after washing with 250 μl washing buffer (0.05% Tween® 20 in PBS, pH 7.4) for 3 min, the wells were blocked with blocking buffer (1% BSA in PBS, pH 7.4) for 1 hour, followed by incubation with the proper amount of samples or recombinant PGRN protein (Human Progranulin DuoSet ELISA, R&D Systems) at concentrations of 62.5, 125, 250, 500, 1000, 2000 and 4000 pg/mL for 2 hours. The wells were then washed three times before incubation with the detection Antibody (Human Progranulin DuoSet ELISA, R&D Systems) for 2 hours. Then the plate was incubated with Streptavidin-HRP solution for 20 minutes at room temperature. After 3 washes, 50 μL of TMB substrate solution (Thermo Fisher Scientific) was added into the wells and the plate was incubated for 5–15 min until blue color appeared. The reaction was stopped with 2N H2SO4 solution (Thermo Fisher Scientific). The optical density was determined by a microplate reader using the readings at 450 nm and subtracting the readings at 540 nm.
3
ELISA-based Quantification of Antigen-Antibody Interactions
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OVA SAPNs
and HrHA SAPNs with 10-fold serial dilutions (10–4–1 ng/μL and 10–6–1 ng/μL,
respectively) were coated onto Maxisorp 96 well immune assay plates
(Nunc) and incubated overnight at 25 °C. The next day, the coated
plates were washed three times with PBS containing 0.1% Tween-20 and
each well was blocked with 1% bovine serum albumin (BSA) in PBS for
1 h at 25 °C. Then, the plates were washed three times and incubated
with HRP-conjugated rabbit anti-OVA antibodies (Thermo Fisher Scientific)
diluted at 1:3,000 for 1 h at 25 °C. For anti-HrHA and anti-4M2e
antibody binding assay, the plates were incubated with anti-HrHA and
anti-4M2e serum antibodies, which were harvested from mice administered
with 2 doses of 20 μg of HrHA-tGCN4 and 20 μg of tZE-4M2e,
diluted at 1:5000 and 1:2500, respectively, for 1 h at 25 °C.
Afterward, plates incubated with the anti-HrHA and anti-4M2e serum
antibodies were washed and incubated with a 1:5000 dilution of HRP-conjugated
goat antimouse IgG (H+L) (SouthernBiotech) for 1 h at 25 °C.
Each well was washed three times and revealed with 1-Step Ultra TMB
substrate solution (Thermo Fisher Scientific) for 20 min. The enzymatic
activity of HRP was stopped by adding 2 N H2SO4 solution (Thermo Fisher Scientific). Absorbance at 450 nm was measured
on a BioTek Synergy H4 Micro plate reader with the correction wavelength
set to 540 nm.
and HrHA SAPNs with 10-fold serial dilutions (10–4–1 ng/μL and 10–6–1 ng/μL,
respectively) were coated onto Maxisorp 96 well immune assay plates
(Nunc) and incubated overnight at 25 °C. The next day, the coated
plates were washed three times with PBS containing 0.1% Tween-20 and
each well was blocked with 1% bovine serum albumin (BSA) in PBS for
1 h at 25 °C. Then, the plates were washed three times and incubated
with HRP-conjugated rabbit anti-OVA antibodies (Thermo Fisher Scientific)
diluted at 1:3,000 for 1 h at 25 °C. For anti-HrHA and anti-4M2e
antibody binding assay, the plates were incubated with anti-HrHA and
anti-4M2e serum antibodies, which were harvested from mice administered
with 2 doses of 20 μg of HrHA-tGCN4 and 20 μg of tZE-4M2e,
diluted at 1:5000 and 1:2500, respectively, for 1 h at 25 °C.
Afterward, plates incubated with the anti-HrHA and anti-4M2e serum
antibodies were washed and incubated with a 1:5000 dilution of HRP-conjugated
goat antimouse IgG (H+L) (SouthernBiotech) for 1 h at 25 °C.
Each well was washed three times and revealed with 1-Step Ultra TMB
substrate solution (Thermo Fisher Scientific) for 20 min. The enzymatic
activity of HRP was stopped by adding 2 N H2SO4 solution (Thermo Fisher Scientific). Absorbance at 450 nm was measured
on a BioTek Synergy H4 Micro plate reader with the correction wavelength
set to 540 nm.
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