N2a/α-SYN and N2a control cells were resuspended in RIPA lysis buffer (Beyotime) containing a protease inhibitor (cocktail, Sigma–Aldrich), lysed on ice for 30 min, and centrifuged at 12,000× g for 15 min. The supernatant protein concentration was detected by a BCA Protein Assay kit (Beyotime). Samples with equal amounts of total proteins (30–50 μg) were subjected to 10 or 12.5% SDS-polyacrylamide gel electrophoresis and then transferred to 0.45 μm polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with the primary antibody at 4°C overnight. Then, the membrane was incubated with a secondary antibody, and the protein bands were detected with the enhanced chemiluminescence detection kit (Wanlei Biotechnology, Shenyang, China) and quantified using the FluorChem Q system (Protein Simple, San Jose, CA, USA) and normalized against reference protein values.
Enhanced chemiluminescence detection kit
Manufactured by Wanlei
Sourced in China
The Enhanced Chemiluminescence Detection Kit is a laboratory equipment used to detect and quantify proteins in Western blot analysis. The kit utilizes a chemiluminescent substrate that emits light when exposed to the enzyme-labeled target proteins, allowing for sensitive and high-resolution detection of protein bands.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Las 4000 system, by Fujifilm (2 mentions)
Bca protein assay kit, by Applygen (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
0.45 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
3 protocols using enhanced chemiluminescence detection kit
1
Western Blot Analysis of α-SYN in N2a Cells
2
Western Blot Analysis of Protein Expression
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HeLa and SiHa cells were lysed in RIPA buffer with protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl uoride) or phosphatase inhibitors (Phosphatase Inhibitor Cocktail, Roche). Protein concentration was detected using BCA protein assay kit (Applygen, Beijing, China). Twenty micrograms of protein was separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Roche Life Science, Basel, Switzerland). After blocking the membrane with 5% non-fat milk for 2 h at room temperature, the targeted proteins were incubated overnight at 4 °C with the following speci c primary antibodies: FTH, TfR1, ferroportin 1 (FPN1), cyclin D1, cyclin E, p-ERK, ERK, p-c-Raf (Ser338), p-MEK1/2, PCNA, NDRG1, c-myc, and β-actin. Thereafter, the membranes were washed and exposed to peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse). Finally, the membranes were detected with an enhanced chemiluminescence detection kit (Wanleibio, Shenyang, China). The total density of the protein bands was detected using the LAS4000 System (FujiFilm).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa and SiHa cells were lysed in RIPA buffer with protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl uoride) or phosphatase inhibitors (Phosphatase Inhibitor Cocktail, Roche). Protein concentration was detected using BCA protein assay kit (Applygen, Beijing, China). Twenty micrograms of protein was separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Roche Life Science, Basel, Switzerland). After blocking the membrane with 5% non-fat milk for 2 h at room temperature, the targeted proteins were incubated overnight at 4 °C with the following speci c primary antibodies: FTH, TfR1, ferroportin 1 (FPN1), cyclin D1, cyclin E, p-ERK, ERK, p-c-Raf (Ser338), p-MEK1/2, PCNA, NDRG1, c-myc, and β-actin. Thereafter, the membranes were washed and exposed to peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse). Finally, the membranes were detected with an enhanced chemiluminescence detection kit (Wanleibio, Shenyang, China). The total density of the protein bands was detected using the LAS4000 System (FujiFilm).
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