The hippocampus was taken out together with the inferior temporal cortex and prefrontal cortex (abbreviated hereafter as cortex). Then, we homogenized the tissue in neuronal Protein Extraction Reagent (Thermo Scientific, 87792) containing a cocktail of protease and phosphatase inhibitors (Thermo Scientific, 87786). Protein concentrations were determined using BCA protein assay (Thermo Scientific, 23227). Equal amounts of total protein were analyzed by 12% SDS-PAGE gels. Then, we transferred it to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies rabbit anti-IDE (Abcam, 1: 1000) were used. Image Lab (Bio-Rad) was utilized for protein band densitometry.
Nc membranes
Manufactured by Solarbio
Sourced in China
NC) membranes are a type of laboratory equipment primarily used for filtration and separation processes. They are made of nitrocellulose and serve as a porous medium for various applications, such as protein or nucleic acid transfer and detection in biochemical analyses.
Automatically generated - may contain errors
Lab products found in correlation
Protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ide, by Abcam (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Cocktail of protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cleaved caspase 3, by Abcam (1 mentions)
Rabbit anti bcl 2, by Abcam (1 mentions)
Rabbit anti bax, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rabbit anti bace1, by Abcam (1 mentions)
Rabbit anti p75ntr, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using nc membranes
1
Hippocampus Protein Extraction and Analysis
2
Hippocampal Protein Analysis Using Western Blot
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Hippocampus tissues were removed and homogenized in neuronal Protein Extraction Reagent (Thermo Fisher Scientific, 87792) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, 87786). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, 23227). Equal amounts of total protein were separated in 12% SDS–PAGE gels and then transferred to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies were: rabbit anti-ADH1B (Biorbyt, 1:800), rabbit anti-BACE 1 (Abcam, 1:1000), rabbit anti-IDE (Abcam, 1:1000), rabbit anti-p75NTR (Cell Signaling Technology, 1:1000), rabbit anti-cleaved caspase-3 (Abcam, 1:1000), rabbit anti-Bcl-2 (Abcam, 1:1000), and rabbit anti-Bax (Abcam, 1:1000). Image Lab (Bio-Rad) was utilized for protein signal densitometry. Detection of proteins from pretreated SH-SY5Y cells using western blotting were performed as previously described (Zhang et al., 2016 (link)).
3
Hypoxia-Induced Protein Regulation
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Cells treated with or without fucoidan under normoxic or hypoxic conditions were collected, washed twice with chilled PBS and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) or using a Nucleoprotein cytoplasm protein extraction kit (KeyGen Biotech, Nanjing, China), according to the manufacturer’s instructions. Equal amounts of protein extract were subjected to 12% SDS-PAGE gels and transferred to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies, including dilution conditions, are as follows: rabbit anti-p-PI3K (1:500), p-Akt (1:500), p-mTOR (1:500), NF-κB (1:500) and anti-His or mouse anti-GAPDH as an internal control for HIF-1α or other proteins.
4
Fucoidan Modulates Apoptosis Pathways
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Western blot was performed to evaluate the effects of fucoidan on the expression of apoptosis factors. Cells treated with different concentrations of fucoidan and Aβ + d -Gal were collected and then washed twice with chilled PBS and lysed in RIPA buffer (Sigma, St. Louis, MO, USA). Equal amounts of protein extract were subjected to 12% SDS-PAGE gels and transferred to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies, including dilution conditions, are as follows: rabbit anti-caspase-3 (1:500), caspase-8 (1:300), caspase-9 (1:1000), cytochrome c (1:200), Livin (1:200), XIAP (1:200), and anti-GAPDH (1:5000) as an internal control for other proteins. Membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:15,000) (Thermo Fisher Scientific) for 1 h at room temperature. HRP-labeled antibodies bound to the membranes were detected by chemiluminescence.
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