Total protein was extracted from cells using a total protein extraction kit according to the manufacturer's instructions (KeyGen, Rockville, MD, USA). The protein concentrations of the lysates were measured with the Protein BCA Assay Kit (Bio-Rad, Hercules, CA, USA). For the Western blotting assay, 20 µg of protein mixed with 5 × SDS loading buffer was loaded per lane, separated with 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in TBST (TBS containing 0.05% Tween-20) for 1 h at room temperature. The membrane was then incubated at 4°C overnight with primary antibodies against TP53 (1:500, abcam), and GAPDH (1:1500, Santa Cruz Biotechnology, Inc.) followed by incubation with goat anti-mouse IgG (1:1000, Beijing Biosynthesis Biotechnology Co., Ltd.) or goat anti-rabbit antibody IgG (1:1000, Beijing Biosynthesis Biotechnology Co., Ltd.) for 1 h at room temperature. ECL Western blotting detection reagents (New England Biolabs, Ipswich, MA, USA) were used to visualize the target proteins, which were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan).
Ecl western blotting detection reagents
Manufactured by New England Biolabs
Sourced in United States
ECL Western blotting detection reagents are a chemiluminescent substrate used for the detection of proteins in Western blot analysis. The reagents produce light in the presence of the enzyme horseradish peroxidase, which is commonly used to label antibodies in Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Bio-Rad (5 mentions)
Peroxidase conjugated secondary antibody, by New England Biolabs (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Anti gapdh antibody, by Boster Bio (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Goat anti mouse igg, by Bioss Antibodies (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
P raf, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Boster Bio (1 mentions)
5 protocols using ecl western blotting detection reagents
1
Western Blot Analysis of TP53 and GAPDH
2
Western Blot Analysis of TET Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from CRC cell lines with RIPA lysis buffer and a proteinase inhibitor. Protein concentrations were measured with a Protein BCA Assay Kit (Bio-Rad, USA). A total of 20 µg of protein, mixed with 2×SDS loading buffer, was loaded per lane. The proteins in the lysates were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). Membranes were incubated at room temperature for 1 hr with 5% skim milk powder to block nonspecific binding. Then the membranes were incubated for 12 hr at 4 °C with an antiserum containing antibodies against TET1, TET2 and TET3 (Santa Cruz, CA). A peroxidase-conjugated secondary antibody (1:1000 dilution) and ECL Western blotting detection reagents were used to visualize the target proteins (ECL New England Biolabs, USA) and the proteins were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan). An anti-GAPDH antibody (Boster, China) was used as a control.
3
Protein Extraction and Western Blot Analysis in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from gastric cancer cell lines using RIPA lysis buffer containing proteinase inhibitor. The protein concentration of the lysates was measured with the Protein BCA Assay Kit (Bio-Rad, USA). For the Western blot assay, 20 µg of protein mixed with 2×SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). To block nonspecific binding, membranes were incubated at room temperature for 1 h with 5% skim milk powder, followed by a 12-h incubation at 4°C with antiserum containing antibodies against c-Src (B-12) (Santa Cruz Biotechnology sc-8056, Santa Cruz, CA, USA), p44/42 MAPK (Erk1/2; Thr202/Tyr204), total ERK, AKT, pRaf, and β-actin (Cell Signaling Technology, Beverly, MA, USA). Ras activity was detected using the Ras Activation Assay Kit from Upstate-Millipore (17–218). A peroxidase-conjugated secondary antibody (1∶5000 dilution) and ECL Western blotting detection reagents (New England Biolabs, USA) were used to visualize the target proteins, which were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan). An anti-β-actin antibody (Boster, Wuhan, China) was used as a protein loading control.
4
Protein Expression Analysis in CRC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from CRC cell lines using RIPA lysis buffer containing a proteinase inhibitor. The protein concentration in the lysates was measured with the Protein BCA Assay Kit (Bio-Rad, USA), and 20 μg of protein mixed with 2 × SDS loading buffer was loaded in each lane. The proteins in the lysates were then separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). To block nonspecific binding, membranes were incubated at room temperature for 1 h in 5% skim milk. Next, the membranes were incubated for 12 h at 4°C with an antiserum containing antibodies against DNMT1, DNMT3A, DNMT3B, SP1, E-cadherin, MGMT or P16, which were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). A peroxidase-conjugated secondary antibody (1:5000 dilution) and ECL western blotting detection reagents were used to visualize the target proteins (ECL New England Biolabs, USA), and the resultant signals were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan). An anti-GAPDH antibody (Boster, China) was used as a protein loading control.
5
Western Blot Analysis of Gastric Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed using standard procedures. Briefly, total protein from the gastric cancer cell lines was extracted using radioimmunoprecipitation assay lysis buffer with a proteinase inhibitor (P0013B; Beyotime Institute of Biotechnology, Haimen, China). The protein concentrations in the lysates were measured with a Protein BCA Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, 30 µg protein per lane was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF (polyvinylidene difluoride) membranes. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.
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!