Total protein was obtained by RIPA lysis buffer (Solarbio, Beijing, China), and the concentration was measured by a BCA assay kit (Yeasen, Shanghai, China). Then 50 μg protein samples was separated on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MD., USA). The membranes were blocked with 5% nonfat milk for 1.5 h and incubated with anti-E-cadherin, N-cadherin, and Vimentin at 4°C overnight. The next day, the membranes were washed and incubated with horseradish peroxidase-labeled IgG antibody at room temperature for 1 h. Images of blots were captured by using the gel imaging system (Bio-Rad, CA., USA), and the original blots are presented in Supplementary file 1 .
Bca assay kit
Manufactured by Yeasen
Sourced in China
The BCA assay kit is a colorimetric method for the quantification of total protein concentration. It uses bicinchoninic acid (BCA) to detect and quantify the amount of protein in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Sds page, by Beyotime (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Anti tubulin, by ABclonal (1 mentions)
P erk1 2, by ABclonal (1 mentions)
Erk1 2, by ABclonal (1 mentions)
E cadherin, by Affinity Biosciences (1 mentions)
Vimentin, by Affinity Biosciences (1 mentions)
P mek1 2, by ABclonal (1 mentions)
Mek1 2, by ABclonal (1 mentions)
N cadherin, by Affinity Biosciences (1 mentions)
Tsg101, by Affinity Biosciences (1 mentions)
Gapdh, by ABclonal (1 mentions)
Ab40776, by Abcam (1 mentions)
Ab131030, by Abcam (1 mentions)
Ab180710, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
6 protocols using bca assay kit
1
Protein Expression Analysis via Western Blot
2
Western Blot Analysis of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from transfected cells using the RIPA lysis and extraction buffer (C1053, PPLYGEN, Beijing, China). Protein concentration was measured using a bicinchoninic acid (BCA) assay kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Total protein (10 µg/lane) was loaded onto SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% skim milk for 1 hour, the membranes were incubated with the primary antibody at 4 °C overnight. The primary antibodies used in this study were mouse polyclonal anti-HA tag (1:2,000, 2063, QB, Wuhan, China) and anti-tubulin (1:1,500, ABclonal Technology, Woburn, MA, USA).
3
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cells and tissues using RIPA buffer, and the concentration of total protein was determined using a BCA Assay Kit (Yeasen Biotech). The protein samples underwent separation using SDS-PAGE and were subsequently transferred to PVDF membranes, followed by blocking in 5% non-fat milk for 2 h. Next, the membranes were incubated with specific primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h. The detection of the band signals was accomplished using an electrochemiluminescence (ECL) system. The following primary antibodies were used in this study TSG101 (Affinity), CD63 (Affinity), N-cadherin (Affinity), E-cadherin (Affinity), vimentin (Affinity), GAPDH (ABclonal), RAS (ABclonal), p-RAF1 (ABclonal), RAF1 (ABclonal), p-MEK1/2 (ABclonal), MEK1/2 (ABclonal), p-ERK1/2 (ABclonal), ERK1/2 (ABclonal), and TPR (Abcam).
4
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 was extracted from cells or tissues using high-performance RIPA lysis buffer (R0010, Solarbio), with the concentration determined using a bicinchoninic acid (BCA) assay kit (Yeasen, Shanghai, China). The proteins from each sample were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) via the wet-transfer method. The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies against FOXM1 (ab180710, 1:1000, Abcam, Cambridge, UK), LMO4 (ab131030, 1:1000, Abcam), AKT (ab8805, 1:500, Abcam), PI3K (ab40776, 1:1000, Abcam), or phosphorylated (p)-PI3K (ab182651, 1:1000, Abcam), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (ab205718, 1:10000, Abcam) at room temperature for 1 h. Development was performed using VILBER FUSION FX5 (Vilber Lourmat, France). The protein bands were quantified using ImageJ 1.48 (National Institute of Health, Bethesda, MD, USA), and normalized to GAPDH. Each experiment was performed in triplicate.
5
Western Blot Analysis of Lung Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Western blot was performed as in a previous study (22 (link)). The lung tissues and cells were collected and added proteinase inhibitor (Vazyme), phosphatase inhibitor (Vazyme), and radio immunoprecipitation assay (RIPA) (Beyotime). Then the mixes were split by using the grinding machine (Jingxin, Shanghai; JXFSTPRP-CL) and centrifuged at 12,000 rpm for 15 min to collect the supernatant. The concentration of supernatant protein was detected by using BCA assay kit (Yeasen). After separation and transfer, the corresponding proteins were measured by using the following antibodies: Nlrp3 (Abcam; ab263899), Phospho-NF-κB (Affinity; AF2006), and Actin (Proteintech; 66009-1-Ig).
6
Protein Expression Analysis in Colorectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW480 and SW620 cells were lysed with lysis buffer (Beyotime). Protein concentration was measured using the BCA Assay Kit (Yeasen). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blockage with 5% non-fat milk, the membranes were cultured with the primary antibodies against USP22 (1:2000, Invitrogen), MMP2 (1:800, Invitrogen), MMP9 (1:1000, Invitrogen) or β-actin (1:5000, Invitrogen) overnight at 4°C. Then, the membranes were cultured with horseradish peroxidase-coupled secondary antibody (1:2000, Invitrogen). Protein signals were observed with an enhanced chemiluminescence (ECL) solution (Beyotime).
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!