Western analyses were performed as described previously44 (link). Briefly, whole cell lysates were extracted using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Mannheim, Germany). The concentration of proteins was determined using enhanced BCA protein assay reagent (Beyotime, Haimen, China) and protein samples were separated by 10–12% SDS-PAGE. Protein was transferred to nitrocellulose and western blot analysis performed. Detection was performed by electro chemical luminescence (ECL).
Enhanced bca protein assay reagent
Manufactured by Beyotime
Sourced in China
The Enhanced BCA Protein Assay Reagent is a colorimetric detection kit used for the quantification of total protein concentrations in biological samples. It is based on the bicinchoninic acid (BCA) method, which provides a linear relationship between protein concentration and absorbance at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Odyssey image station, by LI COR (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Sodium mevalonate, by Merck Group (1 mentions)
Luciferase assay kit, by Promega (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescent kit, by Yeasen (1 mentions)
Kgp113k, by Keygen Biotech (1 mentions)
Enhanced chemiluminescence system, by PerkinElmer (1 mentions)
8 protocols using enhanced bca protein assay reagent
1
Western Blotting Protein Analysis Protocol
2
Mitochondrial Profiling and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial and cytosolic fractions were isolated using the Mitochondria Isolation Kit for Cultured Cells (Pierce, 89874) according to the manufacturer's protocols. For total protein extraction, cells were lysed in RIPA buffer (Beyotime, China, P0013B). Total protein concentration was detected using the Enhanced BCA Protein Assay Reagent (Beyotime, China, P0010), and equal amounts of each sample were boiled with 1× SDS-PAGE sample buffer for 10 min, separated on 8% - 14% SDS-PAGE gels, and transferred to PVDF membranes (Bio-Rad, 162-0177). After blocking with 5% nonfat dried milk, the blots were incubated with the appropriate antibodies and bands were visualized using the enhanced chemiluminescence kit (Bio-Rad, 170-5061). For immunoprecipitation, equal quantities of proteins were incubated with PINK1 antibody at 4°C for 12 h followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003) for 3 h, after which immune complexes were collected by centrifugation. After washing 5 times in phosphate-buffered saline (PBS), samples were subjected to western blot analysis. Densitometric analysis of the blots was performed using Quantity One software (Bio-Rad, Germany).
3
HMF-Mediated Lipid Depletion Impact
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated in 96-well plates at a density of 2.5 × 104 cells/well. After 18 h culture, cells were treated with or without 5, 10, or 20 μmol/L HMF with lipid depletion (LD) medium contained a 1:1 mixture of Ham's F-12 medium (GIBCO, USA) and DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, 5% lipoprotein-deficient serum (Kalen Biomedical, USA), 10 μmol/L compactin (Aladdin), and 50 μmol/L sodium mevalonate (Sigma, USA) for another 18 h. The luciferase activity was measured using luciferase assay kit (Promega, USA) and normalized by the concentration of total proteins using Enhanced BCA Protein Assay Reagent (Beyotime, China).
4
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were lysed with RIPA lysis buffer (P0013B; Beyotime Biotechnology) and detected with Enhanced BCA Protein Assay Reagent (P0010; Beyotime Biotechnology). Each lysate was separated on 10%‐12% SDS‐PAGE gels and then transferred onto PVDF membranes (162‐0177; Bio‐Rad). The membranes were blocked with 5% nonfat dried milk for 2 hours and subsequently incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with a secondary antibody for 2 hours, and then the bands were visualized with the enhanced chemiluminescence kit (170‐5061; Bio‐Rad). The densitometric analysis was measured by Quantity One software (Bio‐Rad).
5
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissue or cells were lysed with lysis buffer and subjected to a 15,000 g spin to remove nuclei and debris. Total protein concentrations were determined using the Enhanced BCA Protein Assay Reagent (Beyotime). Equal amounts (30 μg protein) were loaded onto SDS‐PAGE gels. Proteins were transferred onto polyvinylidene flouride (PVDF) membranes. Membranes were blocked with 5% dry milk solution for 1 hr and then incubated with primary antibodies overnight at 4°C. Membranes were washed with TBST, and protein bands were visualized using horseradish peroxidase‐conjugated species‐specific secondary antibodies. GAPDH was used as loading control. Images were captured, and band intensities were quantified using an Odyssey Image Station (LI‐COR).
6
Western Blot Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with 1% RIPA Lysis Buffer (Beyotime, China). Cell lysate was subjected to a 13,000 rpm spin to remove nuclei and debris. Cleared lysate was used for the indicated biochemical assay. Protein concentrations were determined using the Enhanced BCA Protein Assay Reagent (Beyotime). Equal amounts of cell lysate were loaded onto SDS-PAGE gels and then transferred to PVDF membranes. Membranes were blocked with 5% fat-free dry milk in Tris buffered saline (TBS), containing 0.05% Tween-20, and incubated with primary antibodies. Protein bands were detected by horseradish peroxidase-conjugated species-specific secondary antibodies. Actin was used as loading control. Images were captured using an Odyssey Image Station (LI-COR), and band intensities were quantified using Scion Image.
7
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were lysed with RIPA lysis buffer (Beyotime) supplemented with protease inhibitors and phosphatases inhibitors. Total protein concentration was detected using Enhanced BCA Protein Assay Reagent (Beyotime). Denatured proteins (25 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transfected to PVDF membranes (Keygen Biotech, KGP113K). After blocking with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4°C with the indicated primary antibodies, followed by incubation with a goat anti-rabbit/mouse antibody for 1 h at room temperature. Finally, the protein bands were visualized using the enhanced chemiluminescent kit (Yeasen), and the levels of proteins were normalized to the GAPDH expression.
8
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular samples were lysed with RIPA buffer containing a protease inhibitor cocktail as previously described. Nuclear and cytoplasmic proteins were extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, Shanghai, China) according to manufacturer’s protocol. The protein concentration was determined using Enhanced BCA Protein Assay Reagent (Beyotime, Jiangsu, China). Equal quantities of proteins from each sample were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Bio-Rad, 162–0177). After blocking with 5% fat-free dry milk, the membranes were incubated with specific primary antibodies overnight at 4 °C and corresponding HRP-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized with an enhanced chemiluminescence system (Perkin-Elmer Life Sciences, Boston, USA). Band intensities were quantified using ImageJ software, and β-actin, LaminB1 or GAPDH was used as internal reference.
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!