Cells were seeded at the appropriate density, and total protein of cells or tissues were using cell lysis buffer containing (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100)containing protease inhibitor cocktail (sigma, P2714-1BTL) on ice for 30 mins followed by centrifugation. Protein concentrations was determined with a Bicinchoninic Acid Assay Kit (Beyotime Biotechnology). Protein (20 μg/lane) was separated by 10–15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked for 1.5 hrs in Tris‐buffered saline and Tween 20 (TBST, pH 7.6) containing 5% non‐fat milk powder at room temperature and incubated with primary antibodies at 4°C overnight. The membranes were then washed three times with TBST for 15 mins each and incubated with anti‐rabbit secondary antibodies (1:5,000 in TBST) conjugated to HRP for 1 hr at room temperature. The blots were then developed in the dark by using an ECL detection kit (Proteintech). Band intensities were quantified by ImageJ 1.45 software (NIH, USA). Antibodies are listed in Table 3
Ecl detection kit
Manufactured by Proteintech
Sourced in United States
The ECL detection kit is a laboratory tool used to detect and visualize proteins on Western blot membranes. It utilizes a chemiluminescent reaction to generate a light signal that can be captured and analyzed using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Rab3a, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Membrane and cytosol protein extraction kit, by Beyotime (1 mentions)
Whole cell lysis assay, by Keygen Biotech (1 mentions)
Protease inhibitor phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Wet transfer system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa reagent, by Beyotime (1 mentions)
Abs152589, by Absin (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
6 protocols using ecl detection kit
1
Comprehensive Western Blot Analysis
2
Western Blot Analysis of Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The selected cells were washed with 0.01 M PBS (pH 7.4), harvested, and lysed on ice for 20-30 min using 100 μL of radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). Protein was quantified using a BCA Protein Assay Kit (Pierce, USA) according to the manufacturer’s instructions. Protein samples (60 mg per well) were separated by electrophoresis in 12% SDS-polyacrylamide gels and then blotted onto a 0.22-μm polyvinylidene fluoride (PVDF) membrane (Thermo Scientific) using a Bio-Rad wet transfer instrument. The membranes were blocked overnight in blocking buffer and then probed with antibodies against Rab3a (Abcam, 1:2000), β-actin (Abcam, 1:10000), and PHEV (a lab-prepared monoclonal antibody against the PHEV nucleocapsid [N] protein, 1:500) at 4°C overnight. After washing with 0.1% Tween-20/PBS, the membranes were incubated with the corresponding secondary IgG antibody for 1 h at 37°C. The signal was read using an ECL detection kit (Proteintech, USA).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein and membrane protein from cells was extracted using a whole cell lysis assay (Nanjing Keygen Biotech Co. Ltd., Nanjing, China) and a Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China), respectively. Protein concentrations were determined using the Bicinchoninic Acid Assay (Beyotime Biotechnology, Shanghai, China). The proteins were separated by 8-10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked for 1 h in Tris-buffered saline and Tween 20 (TBST, pH 7.6) containing 5% nonfat milk powder at room temperature and probed at 4°C overnight with primary antibodies for FFA1 (1 : 500 dilution, Santa Cruz Biotechnology, USA), PDX-1 (1 : 1000 dilution, Abcam, USA), BETA2/NeuroD (1 : 500 dilution, Proteintech, USA), and β-actin (1 : 1000 dilution, Proteintech, USA). The membranes were then washed three times with TBST for 15 min each and incubated with anti-rabbit secondary antibodies (1 : 5000 in TBST) conjugated to horseradish peroxidase for 1 h at room temperature. The blots were then developed in the dark by using the ECL detection kit (Proteintech). Band intensities were quantified by ImageJ 1.45 software (NIH, USA) and normalized with β-actin as the internal control.
4
Western Blot Analysis of Caskin1 and PHEV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells in 6-well plates or brain tissues were washed once with phosphate buffer saline (PBS), followed by lysis using a Radio Immunoprecipitation Assay (RIPA) Lysis Buffer and a Phenylmethanesulfonyl fluoride protease inhibitor (Beyotime) on ice for 30 min. The concentration of protein was determined by the BCA Protein Assay kit (Pierce). The protein samples (50 mg/lane) were separated using a 10% polyacrylamide gels and were transferred to 0.22 μm polyvinylidene fluoride membranes using the Bio-Rad wet transfer system. After blocking overnight at 4°C with 5% non-fat dry milk in PBS, the membranes were probed with antibodies against Caskin1 (Synaptic Systems, Göttingen, 1:2000), β-actin (Proteintech, USA, 1:2000) and PHEV (a laboratory-prepared polyclonal antibody to PHEV, 1:500) with an overnight incubation at 4°C. Next, the membranes were washed with PBS containing tween-20 (PBST) four times and were incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse IgG antibodies (Proteintech) for 1 h at 37°C. After washing with PBST, the signal was visualized using an ECL detection kit (Proteintech). β-actin was used as a loading control.
5
Western Blot Analysis of PIMREG Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the protein expression of PIMREG in cells and BC tissues, a western blot assay was performed based on a previously published protocol (Xie et al., 2022 (link)). Briefly, the cells or BC tissues were lysed using RIPA reagent (Beyotime, Shanghai, China) and boiled for 10 min. Subsequently, the total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% nonfat milk diluted with Tris-buffered saline-Tween (TBST) at room temperature for 1 h and incubated overnight at 4 °C with the primary antibody: anti-PIMREG (1:500; abs152589; Absin Bioscience, Inc., Shanghai, China). The next day, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 1 h. The Western blots were visualized using an ECL detection kit (Proteintech Group, Inc., Rosemont, IL, USA) for chemiluminescence. The density of the protein bands was quantified using Image J software (Pierce, Rockford, IL, USA), and the values was normalized to β-Actin.
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment, the cells were lysed in pre-cold RIPA buffer (Beyotime, Shanghai, China) with protease inhibitor PMSF (1:100) and phosphatase inhibitor cocktail (1:100) at 0 °C for 30 min. The protein concentration of each sample was measured using BCA protein assay kit (Beyotime). The protein samples were added into 5 × loading buffer and subjected to heat denaturation at 100 °C for 5 min. Then, the protein (40 μg) was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Following blocking with TBST (Tris-buffered saline with 0.1% Tween-20) containing 5% nonfat milk at room temperature for 1 h to block nonspecific binding, the membranes were incubated with primary antibodies at 4 °C overnight. Then, the membranes were washed with TBST three times and incubated with corresponding second antibodies (horseradish peroxidaseconjugated goat anti-rabbit/mouse antibodies, Proteintech, Chicago, IL, USA) at a dilution of 1:2000 at room temperature for 1 h. After being washed with TBST for three times, the protein-antibody complexes were visualized using an enhanced chemiluminescent (ECL) detection kit (Menlo Park, CA, USA). The images were quantified by Quantity One software (Bio-Rad).
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!