Protein extraction from fresh frozen mouse forebrain (0.4 g) was carried out in 1 mL of 1X TBS containing a protease and phosphatase inhibitors. The tissue was homogenized with Micropestle (Eppendorf, Hamburg, Germany) followed by sonication. The homogenate was centrifuged for 30 minutes at 14,000g at 4 °C. The supernatant (soluble fraction) was transferred into fresh tubes and kept at −80 C until further use. The remaining pellet was re-suspended into 1 ml PBS containing 1% Triton-X followed by sonication and centrifugation as previously mentioned. For the Aβ ELISA, the tissue was homogenized in PBS and then centrifuged at 14,000 rpm for 25 min at 4C. The supernatant was reserved (soluble fraction) and the pellet was homogenized in 0.5M Guanidine, allowed to solubilize for 3 hrs, and then spun at 14,000 rpm for 25 min (insoluble fraction). A sandwich ELISA was performed using antibodies kindly donated by Dr. David Holtzman. Briefly, Nunc Maxi Sorp (Thermo) plates were coated with either HJ2 (anti-Aβ40) or HJ7.4 (anti-Aβ42) and incubated overnight at 4C. Plates were then incubated with blocking buffer for 1.5 hrs. Plates were incubated overnight with the soluble and insoluble tissue samples. The plates were then incubated with HJ5.1 Biotin, which recognizes both forms of Aβ. The plates were coated with Strep-poly-HRP40 (Pierce), and then developed with SuperSlow TMB (Sigma).
Super slow tmb
Manufactured by Merck Group
Sourced in United States
Super Slow TMB is a laboratory reagent used in immunoassays. It is a substrate solution that produces a colored reaction when exposed to horseradish peroxidase (HRP), an enzyme commonly used as a label in various immunoassay techniques. The solution is designed to generate a slow color change, allowing for more precise quantification and extended reaction times.
Automatically generated - may contain errors
Lab products found in correlation
Synergy 2, by Agilent Technologies (2 mentions)
Synergy 2 plate reader, by Agilent Technologies (2 mentions)
Streptavidin poly hrp 40, by Biosynth (2 mentions)
Micropestle, by Eppendorf (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Coomassie staining, by Merck Group (1 mentions)
Las 300 imager, by Fujifilm (1 mentions)
Biotinylated bt2, by Thermo Fisher Scientific (1 mentions)
9 protocols using super slow tmb
1
Extraction and Quantification of Amyloid-Beta
2
Quantification of Aβ40 and Aβ42 in CSF
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Aβ40 and Aβ42 levels from CSF samples were assayed using sandwich ELISAs as previously described (Bero et al., 2011 (link); Roh et al., 2012 (link)). Briefly, Aβ40 and Aβ42 were quantified using monoclonal capture antibodies (a generous gift from David Holtzman) targeted against amino acids 45–50 (HJ2) or 37–42 (HJ7.4), respectively. For detection, both Aβ40 and Aβ42 used a biotinylated monoclonal antibody against the central domain (HJ5.1B), followed by incubation with streptavidin-poly-HRP-40. Assays were developed using Super Slow TMB (Sigma) and the plates read on a Bio-Tek Synergy 2 plate reader at 650 nm.
3
Quantifying Aβ40 in APP/PS1 Mice
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ISF samples from 3-month-old APP/PS1 mice (n = 5) collected from in vivo microdialysis experiments were analyzed for Aβ40 using sandwich ELISAs as previously described (Bero et al., 2011b (link); Roh et al., 2012 ; Macauley et al., 2015 (link)). Briefly, Aβ40 and Aβ42 were quantified using monoclonal capture antibodies (generous gifts from Dr. David Holtzman, Washington University) targeted against amino acids 33–40 (HJ2, Aβ40) or amino acids 35–42 (HJ7.4, Aβ42). For detection, a biotinylated monoclonal antibody against the central domain amino acids 13–28 (HJ5.1B) was used, followed by streptavidin-poly-HRP-40. Assays were developed using Super Slow TMB (Sigma). Plates were read on a Bio-Tek Synergy 2 plate reader at 650 nm.
4
Quantifying Aβ40 in APP/PS1 Mice
5
Quantifying Hippocampal Aβ Levels in APP/PS1 Mice
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Hippocampal ISF samples from 3‐month and 9‐month APP/PS1 mice and WT mice (n = 4–6/group) collected from microdialysis experiments were analysed for Aβ40 and Aβ42 using sandwich ELISAs as previously described (Bero et al., 2011 (link), Cirrito et al., 2003 (link), Cirrito et al., 2005 (link), Macauley et al., 2015 (link)). Briefly, Aβ40 & Αβ42 were quantified using monoclonal capture antibodies (generous gifts from Dr. David Holtzman, Washington University) targeted against amino acids 33–40 (HJ2) or 35–42 (HJ7.4) respectively. For detection, a biotinylated monoclonal antibody against the central domain amino acids 13–28 (HJ5.1B) was used, followed by streptavidin‐poly‐HRP‐40 (Fitzgerald). The assay was developed using Super Slow TMB (Sigma) and the plates were read on a Bio‐Tek Synergy H1 plate reader at 650 nm. Standard curves with known Aβ standards related optical density measures to Aβ concentrations. Statistical significance was determined using a one‐way ANOVA. Data is represented by means ± SEM.
6
Quantifying Aβ1-x Levels in APP/PS1 Mice
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ISF samples from Kir6.2+/+, APP/PS1 mice, Kir6.2-/-, APP/PS1 mice, and Kir6.1-/-, APP/PS1 mice (n=7-8/group) collected from in vivo microdialysis experiments were analyzed for Aβ1-x using sandwich ELISAs as previously described [14, 39] . Briefly, Aβ1-x was quantified using a monoclonal capture antibody targeted against Aβ13-28 (m266) and a biotinylated detection antibody targeted against Aβ1-5 (3D6), both generous gifts from Dr. Ron DeMattos, Eli Lilly and Co., Indianapolis, IN. After incubation with streptavidin-poly-HRP-20, the assay was developed using Super Slow TMB (Sigma) and the plates read on a Bio-Tek Synergy 2 plate reader at 650nm. Statistical significance was determined using a two-tailed, unpaired t-test. Data is represented by means +/-SEM.
7
Recombinant Mycoplasma Pneumoniae Protein Analysis
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The result of expression of recombinant proteins rPDHA-D was analyzed by SDS-PAGE using 10% NuPage Bis-Tris gels (Life, Darmstadt, Germany, #NPO303 Box) followed by Coomassie staining (Merck, # 115444). To confirm the specificity of the produced polyclonal antisera, blotted M. pneumoniae whole cells were incubated with the antisera to rPDHA-D (1:500) and detected with anti-guinea pig IgG (1:750; Dako, Hamburg, Germany, #P01402-2). Chemiluminescence signals were digitally recorded using a LAS-300 imager (Fujifilm, Düsseldorf, Germany).
To compare the quantitative reactivity of sera by ELISA, cavities of 96-well plates (Greiner, #655001) were coated overnight with M. pneumoniae total proteins (15 μg/ml) and blocked as described [21 (link)]. Wells were incubated with guinea pig sera to whole proteins of M. pneumoniae and to subunits of the PDH complex (1:250) followed by anti-guinea pig IgG (1:750). After incubation with the substrate (TMB super slow, Sigma, #T5569), the reaction was stopped with 1 M HCl and absorbance was detected at 450 nm with reference wavelength of 620 nm.
To compare the quantitative reactivity of sera by ELISA, cavities of 96-well plates (Greiner, #655001) were coated overnight with M. pneumoniae total proteins (15 μg/ml) and blocked as described [21 (link)]. Wells were incubated with guinea pig sera to whole proteins of M. pneumoniae and to subunits of the PDH complex (1:250) followed by anti-guinea pig IgG (1:750). After incubation with the substrate (TMB super slow, Sigma, #T5569), the reaction was stopped with 1 M HCl and absorbance was detected at 450 nm with reference wavelength of 620 nm.
8
Tau-5 Antibody ELISA Assay
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Tau-5 antibody (0.5 μg/well) was coated in wells of 96 half well plates. On the next day, the plates were blocked with 4% BSA/PBS for 1 h at 37°C. Samples and standards diluted with ELISA sample buffer (0.25% BSA/PBS supplemented with complete) were applied and incubated at 4°C overnight. On the next day, biotinylated HJ8.7 (a kind gift from Dr. Holtzman) or biotinylated BT2 (Thermo Fisher Scientific) were applied at the concentration of 0.3 μg/ml and incubated at 37°C for 1.5 h, followed by incubation with streptavidin-poly-HRP40 (Fitzgerald) at room temperature for 1.5 h. Assays were developed with TMB superslow (Sigma) and the reaction was stopped by addition of 1M H3PO4 and read at 450 nm.
9
ELISA Quantification of Aβ40 and Aβ42 in 5xFAD Mice
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