Transfected cells were lysed in RIPA buffer containing 1% protease inhibitor AEBSF (LanMu, Shanghai, China) for 30 min, centrifuged at 12,000 rpm for 15 min at 4 °C, and the protein concentrations were determined using the Pierce Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Total proteins were separated by SDS-PAGE (the concentration was based on the molecular weight of the antibody), and transferred to polyvinylidene fluoride (PVDF) membranes (0.2 μm, Merck KGaA, Darmstadt, Germany). Membranes were blocked in the buffer containing 5% non-fat milk in Tris-buffered saline, containing 0.1% Tween-20 (TBST) for 1h and incubated overnight with the following primary antibodies: rabbit monoclonal anti-TGFβRI (ab135814) (1:100), anti-SMAD3 (ab40854), anti-p-SMAD3 (phospho S423 + S425) (ab52903), anti-RAS (ab52939), anti-p53 (ab75754), and anti-TGF-βI (ab179695) (1:1000) at 4 °C. Membranes were washed with TBST, and incubated with goat anti-rabbit IgG H&L (ab97047) secondary antibodies (1:30,000). A rabbit polyclonal to β-actin antibody (ab119716) was used as a loading control. The straps were detected by an Odyssey Infrared Imaging System and relative protein levels of the genes involved were measured by the gray-scale value of straps using Image J software.
Pierce bicinchoninic acid bca protein assay kit
Manufactured by Beyotime
Sourced in China
The Pierce bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantification method for total protein concentration. It utilizes the reduction of Cu2+ to Cu+ by protein in an alkaline medium, and the selective colorimetric detection of the Cu+ ion using a reagent containing bicinchoninic acid.
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4 protocols using pierce bicinchoninic acid bca protein assay kit
1
TGF-β Signaling Pathway Protein Analysis
2
Western Blot Analysis of Protein Markers
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The cells were lysed in RIPA buffer containing the protease inhibitor AEBSF (1% w/v) (LanMu, Shanghai, China) for 30 min, centrifuged at 12,000 rpm for 15 min at 4°C, and the protein concentrations were determined using the Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Total proteins were separated by SDS-PAGE (the acrylamide concentration was based on the molecular weight of the protein of interest) and transferred to polyvinylidene fluoride (PVDF) membranes (0.2 µm, Merck KGaA, Darmstadt, Germany).36 (link) The membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 (TBST), containing 5% non-fat milk, for 1h and incubated overnight with the following primary rabbit monoclonal antibodies (Abcam): anti-SERTAD (ab107728 at a dilution of 1/100), anti-p21 (ab109520; 1/1000), anti-p38 (ab170099; 1/1000) and anti-p53 (ab75754; 1/1000) at 4°C. Membranes were washed with TBST and incubated with goat anti-rabbit IgG H&L (DyLight™800 4X PEG Conjugate) (Cell Signaling Technology 5151S; dilution: 1/30,000) secondary antibodies. A rabbit polyclonal anti-GAPDH antibody (ab8245; dilution: 1/1000) was used as a loading control. The bands were detected by an Odyssey Infrared Imaging System and relative levels of protein expression were determined according to the gray-scale value of the bands using Image J software.
3
Cytokine Quantification in Cell and Tissue Samples
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IL-4, IL-10, IL-13, TNF-α, and IL-6 ELISA kit were purchased from Jiangsu Meimian industrial Co., Ltd, China. Cells were cultured for 24 h in DMEM with 10% FBS, and the supernatant of both cell types was then collected to measure the levels of IL-4, IL-10, IL-13, TNF-α, and IL-6 according to the manufacturer’s instruction. As for tissue sample, 1 cm-long spinal cord (cells grafts were at the center of the spinal cord) was lysed with RIPA Lysis Buffer and then quantified with Pierce bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Thereafter, the tissue supernatant was prepared for ELISA using their respective kits according to the manufacturer instructions.
4
Cytokine Profiling of 3D and 2D HPMSCs
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Cells and tissue samples were assayed using and Bio-Plex ProTM human cytokine 27-plex assay kit (Bio-Rad#10014905) according to the manufacturer’s instructions. As for cells sample, cell supernatant from 3D- and 2D-HPMSCs at a density of 105/ml were collected after appropriate culture. As for tissue sample, 1cm-long spinal cord (cells grafts were at the center of the spinal cord) was lysed with RIPA Lysis Buffer and then quantified with Pierce bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). All the cells and tissue supernatant were next prepared for assay. Data was processed using Bio-Plex Manager software version 6.1 (Bio-Rad Laboratories, USA).
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