Three metastatic colon adenocarcinoma tissues (pathologic stage: pT3N1Mx) were mixed as the Meta (M) protein sample, and three age matched primary colon adenocarcinoma tissues (pathologic stage: pT3N0Mx) were mixed as Colon (C) protein sample. The equal-load amount of C and M samples were used for Western blotting analysis. Anti-CDK1 antibody (Abcam, United States) was used to detect the protein level of CDK1. Signals were detected with Immobilon ECL reagents (Millipore, United States).
Immobilon ecl reagent
Manufactured by Merck Group
Sourced in United States
Immobilon ECL reagent is a chemiluminescent detection system designed for the sensitive and quantitative detection of proteins on Western blots. It utilizes a proprietary luminol-based substrate that produces a strong, stable light emission when combined with the appropriate enzyme-conjugated detection antibodies.
Automatically generated - may contain errors
7 protocols using immobilon ecl reagent
1
CDK1 Expression in Metastatic Colon Cancer
2
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were mixed with an equal volume of 2X Laemmli sample buffer, boiled for 5 min, loaded on a SDS-gel, and then transferred to a PVDF membrane for Western blot, as detailed previously in [16] (link). After blocking for one hour in 5% dry milk, the membrane was incubated with primary antibody (1–2 µg) in TBS containing 0.1% Tween-20 and 5% bovine serum albumin for 90 min at room temperature followed by extensive rinses and one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1∶10,000). Chemiluminescence signals were developed using Millipore Immobilon ECL reagents.
3
Drosophila Embryo Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Assays were performed with extracts prepared from Drosophila embryos that were lysed in RIPA buffer (50 mM Tris–HCl (pH 8),150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate,1% Triton X-100, 1 mM PMSF, and protease inhibitors (cOmplete Tablets, Roche)). Extracts were immunoprecipitated using anti-Kkv antibodies or a mock antibody (anti-CP190), followed by incubation with Protein G Dynabeads (Invitrogen). Immunoprecipitates were washed with RIPA buffer and analysed by western blot using anti-Kkv or anti-Reb antibodies and the Immobilon ECL reagent (Millipore). Original uncropped blots can be found in the S1 Raw Images.
4
Quantitative Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting analysis was conducted as previously described 33 (link). Compound-treated cells were resuspended in RIPA lysis buffer. After centrifugation at 16,200 g for 10 min at 4 °C, supernatants were collected in a pre-cooled microfuge tube. Protein concentration was determined by the BCA Protein Assay. Equal amounts of protein were loaded into 10% SDS-PAGE, and proteins were further transferred onto PVDF membranes after electrophoresis. Membranes were blocked with 5% FBS in TBST for 1 h at room temperature followed by incubation with primary antibodies, including antibodies against CDK4, CDK6, β-actin (Santa Cruz Biotechnology, sc-23896, sc-7961, sc-8432), BCL-2 (BD Biosciences, 560637), cyclin D1, p-Rb, Rb, Ac-Histone H3, caspase-3, p-JAK1, JAK1, p-STAT3 and STAT3 (Cell Signaling Technology, CST# 2978, 8516, 9309, 8173, 9662, 3331, 3332, 9145, 4904) overnight at 4 °C. Membranes were washed with TBST three times followed by incubation in HRP-labelled secondary antibodies for 1 h at room temperature. Signal was detected using Millipore Immobilon ECL reagent.
Protein signal density was quantified using ImageJ software (Version 1.60, National Institutes of Health, USA) and subsequently labelled under the bands. All lanes were normalized by dividing the density by the density of β-actin.
Protein signal density was quantified using ImageJ software (Version 1.60, National Institutes of Health, USA) and subsequently labelled under the bands. All lanes were normalized by dividing the density by the density of β-actin.
5
Detecting Digoxigenin-Labeled Nucleic Acids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digoxigenin-labeled RNAs and DNAs prepared by in vitro transcription (Makeyev et al., 2007 (link)) or nick translation (Yap et al., 2018 (link)), respectively, were spotted onto 0.45-μm nitrocellulose membrane (Sigma Aldrich, cat# GE10600016) and UV crosslinked at 120 mJ/cm2 (Stratalinker). The membrane was rinsed with 1 × TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween 20) and blocked with 5% BSA in 1 × TBST at room temperature for 1 hour. It was subsequently incubated with HyPro protein diluted in 1 × TBST and 1% BSA at room temperature for 1 hour followed by three washes with 1 × TBST. The membrane was then soaked in reconstituted Immobilon ECL reagent (Millipore, cat# WBKLS0500) and imaged using an Odyssey FC system (LI-COR).
6
Drosophila Salivary Gland Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Assays were performed with extracts prepared from salivary glands of Drosophila third-instar larvae that were lysed in RIPA buffer (50 mM Tris-HCl pH8,150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate,1% Triton X-100, 1 mM PMSF and protease inhibitors (cOmplete Tablets, Roche, 04693159001). Extracts were immunoprecipitated using αFlag antibodies or a control antibody (αAbd-B), followed by incubation with Protein G Dynabeads (Invitrogen, 10003D). Immunoprecipitates were washed with RIPA buffer and analysed by Western blot using either αBtl, αV5, DCAD2 or αFlag antibodies and the Immobilon ECL reagent (Millipore, WBKLS0100). Uncropped and unprocessed scans of blots are provided in the Source Data file or in the Supplementary Information.
7
Western Blot Analysis of TR146 Cells
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!