Treated/infected THP-1 macrophages were lysed using a RIPA protein extraction protocol, then Smad3 levels within the cell lysates was determined using a SMAD3 (Phospho) (pS423/pS425) Human InstantOne™ ELISA Kit (catalog # 85-86192-11) per the manufacturer’s instruction (ThermoFisher, Waltham, MA). Results were assessed using a Multiskan FC plate reader (Fisher Scientific, Waltham, MA) read at 450 nm. All sets were performed in triplicates.
Human instantone elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human InstantOne™ ELISA Kit is a laboratory reagent designed for the quantitative measurement of target analytes in human samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the analyte of interest. The kit provides the necessary reagents and components to perform the ELISA assay.
Automatically generated - may contain errors
4 protocols using human instantone elisa kit
1
Quantification of Phosphorylated Smad3
2
Quantifying Phosphorylated NF-kB p65
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NF-kB activity was evaluated in cell lysates using the commercial ELISA kit NF-kB p65 (Phospho) [pS536] Human InstantOne™ ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA). This kit is specifically engineered for the measurement of phosphorylated (pS536) human p65. Briefly, 50μL of sample lysate, a negative control and a positive control were added to the plates and mixed with 50 μL of the antibody cocktail and incubated for 1h at room temperature. After washing, 100 μL of the detection reagent was added to each well and incubated within 20 min with shaking at 300 rpm. Finally, the reaction was stopped by adding 100 μL of stop solution. The absorbance of each sample was measured using a spectrophotometric microplate reader (DAS, Italy; IT) set at λ450 nm. Values are expressed as the relative optical density (OD) per mg protein.
3
Quantifying NFκB and TGFβ1 in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate NFκB p65, lymphocytes (5 × 10 4 cells/well) were lysed, and total and phosphorylated (Ser536) NFκB p65 were measured using a NFκB p65 (Total/Phospho) Human InstantOne™ ELISA Kit (Thermo Fisher Scientific), according to the manufacturer's protocol.
To evaluate TGFβ1, tumor cells were seeded into a 6-well plate. After 48 h of incubation, supernatants were collected, and the concentration of TGFβ1 was measured using the Human TGFβ1 ELISA Kit (Proteintech, USA) according to the manufacturer's protocol.
To evaluate TGFβ1, tumor cells were seeded into a 6-well plate. After 48 h of incubation, supernatants were collected, and the concentration of TGFβ1 was measured using the Human TGFβ1 ELISA Kit (Proteintech, USA) according to the manufacturer's protocol.
4
Quantifying SMAD1/5/8 Pathway Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We assayed the activation status of the SMAD1/5/8 pathway by using the SMAD1[pS463/pS465] Human Instan-tOne ELISA Kit (no. 85-86182-11, ThermoFisher Scientific). Briefly, 900,000 cells expressing each of the BMPR2 variants were seeded in a 6-well plate and cultured in RPMI-1640 media supplemented with 5% fetal bovine serum and 1 mg/mL of doxycycline. When indicated, cells were treated with 50 ng/mL BMP4 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 minutes and processed according to the manufacturer's instructions. Five micrograms of each protein extract were assayed in a single step, as the sandwich formation and adsorption to the assay plate are performed simultaneously. Phospho-SMAD1 levels between BMP4-stimulated and nonstimulated conditions were quantified.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!