Reagents were purchased from Sigma (St. Louis, MO) unless otherwise specified. The UltraLink hydrazide resin, Zeba spin desalting columns, Pierce centrifuge columns, radioimmuno-precipitation assay (RIPA) buffer, and chemiluminescence substrate kit were from Thermo Scientific (Rockford, IL). Sequencing-grade trypsin was from Promega (Madison, WI). The YM-30 kDa and YM-50 kDa MWCO centrifugal filters and C18 ZipTips were from Millipore (Billerica, MA). The 200 mesh Formvar/carbon-coated grid was from Electron Microscopy Sciences (Hatfield, PA). The pooled normal human serum sample was obtained from Innovative Research (Novi, MI). The monoclonal anti-CD9 antibody (no. ab92726) and horseradish peroxidase (HRP) conjugated secondary antibody were from Abcam (Cambridge, MA). The 4–20% SDS-PAGE gel was from Bio-Rad (Hercules, CA), and the ProteoSilver Plus Silver Stain Kit was from Sigma. The phosphate-buffered saline (PBS) buffer and 1 M NaCl were filtered with a 0.22 μm filter prior to use.
Chemiluminescence substrate kit
Manufactured by Thermo Fisher Scientific
The Chemiluminescence substrate kit is a laboratory product designed to facilitate the detection and quantification of proteins using chemiluminescence techniques. The kit provides the necessary reagents and substrates to enable the chemiluminescent reaction, allowing for sensitive and accurate protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ibright imaging system, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Proteosilver plus silver stain kit, by Merck Group (1 mentions)
Pierce centrifuge column, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Zfp36l2, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Image studio lite software version 5, by LI COR (1 mentions)
Alx fpr2, by Abcam (1 mentions)
Gpr32, by Abcam (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
6 protocols using chemiluminescence substrate kit
1
Proteomic Analysis of Extracellular Vesicles
2
Protein Expression Analysis of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein sample from cell lines was extracted with RIPA lysis buffer (Sigma-Aldrich, USA) and corresponding protein concentration was determined with a BCA protein assay kit (Beyotime). Equal amount of protein sample was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against ZFP36L2, E-cadherin, N-cadherin, Vimentin and GAPDH (Abcam, Cambridge, MA, USA) overnight at 4 °C. Following incubation with corresponding secondary antibodies for 2 h at room temperature, the protein bands were visualized with Chemiluminescence Substrate Kit (Thermo Fisher Scientific).
3
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells plated on 6-well plates were lifted with 2 mM EDTA-PBS, collected in tubes, and pelleted (1000 rpm for 5 min). Cell pellets were lysed in RIPA lysis buffer (Thermofisher Scientific, #89900) supplemented with 1% protease inhibitor (Calbiochem #539131) and stored at -80°C until further analysis. Protein concentration was determined using a BCA Protein Assay Kit (Thermofisher Scientific, #23225). Equal amounts of total protein samples were electrophoretically separated onto 4% to 20% Tris-glycine gels (Invitrogen, #XP04205BOX), then transferred to nitrocellulose membranes, blocked for 1 hour at room temperature in Tris-buffered saline with Tween 20 (TBST; 10 mmol/L Tris-HCl buffer, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% BSA, and hybridized with appropriate primary antibodies overnight at 4°C. After four washes in TBST, membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature, then washed again four times in TBST prior to development with a chemiluminescence substrate kit (Thermofisher Scientific, #34580). Images were taken on an iBright imaging system (iBright imaging system; Thermofisher Scientific). Protein expressions were analyzed using ImageJ software and normalized to β-Actin or GAPDH. Two to eight experimental repeats were performed for each experiment. Representative blots are shown for each protein.
4
Western Blot Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of total proteins were loaded onto 4–20% Tris–glycine gels (Invitrogen, #XP04205BOX) and transferred to polyvinylidene fluoride (PVDF) membranes. All membranes were blocked in Tris-buffered saline with Tween 20 (TBST; 10 mmol/L Tris–HCl buffer, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween 20) with 5% bovine serum albumin (BSA) at RT for 1 h, followed by overnight incubation with primary antibodies (see antibody details and dilutions in Table 1 ). The membranes were then washed in TBST four times before secondary antibody incubations for 1 h, 30 min at RT (see antibody details and dilutions in Table 1 ). After final washing in TBST four times, the proteins were exposed using a chemiluminescence substrate kit (Thermo Fisher Scientific, #34,580), and images were obtained using the iBright imaging system (iBright imaging system; Thermo Fisher Scientific). Image Studio Lite software version 5.2 (LI-COR Biosciences, Lincoln, NE) was used to analyze protein expression. One representative blot is shown for each molecule.
5
Western Blot Analysis of Signaling Proteins
6
Evaluating Apoptosis and Metastasis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were obtained from the transfected cervical cancer cells using Laemmli S.D.S reducing buffer (2% S.D.S, 50 mM Tris–HCl, and 10% glycerol; pH 6.8) treatment. The protein lysates were separated on 8%–10% polyacrylamide gel and blotted to PVDF membrane. Antibodies against Bax, Bcl-2, MMP-2, MMP-9, RAB2B (Abycam) and β-actin (Sigma-Aldrich) were used to treat PVDF membranes. The membranes were treated with horseradish peroxidase-conjugated secondary antibodies and specific protein bands were detected using Chemiluminescence Substrate Kit (Thermo Fisher Scientific).
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!