A459 and PC9 cells were trypsinized, centrifuged, and harvested after 120 h of treatment. The cell contents were collected after cell lysis, and total soluble protein was collected by centrifugation. The protein concentration was determined using the BCA (bicinchoninic acid) method. After the protein concentration was determined, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed for each tissue/cell. Following electrophoresis, the proteins separated by electrophoresis were transferred to polyvinylidene fluoride (PVDF) membranes using a transfer device (millipore, USA). After transfer, the PVDF membrane was removed from the transfer device, blocked with 5% skim milk for 1 hour, and rinsed with PBS. Next, primary antibodies against EFNA3 (1:500), Bax (1:500), and caspase3 (1:500) were added, and the membranes were incubated at 4 ℃ overnight. Subsequently, a secondary goat anti-rabbit antibody (1:1,000) was added, and the membranes were incubated for 1 h. A chemiluminescence imaging system (Thermo Fisher SCIENTIFIC, USA) was used to expose and visualize the protein on the PVDF film.
Chemiluminescence imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Chemiluminescence Imaging System is a laboratory instrument designed to capture and analyze chemiluminescent signals. It uses a sensitive camera to detect and quantify light emission from chemiluminescent samples, enabling the visualization and analysis of various biological and chemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Piercetm ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membranes 0.45 μm, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
A1011, by ABclonal (1 mentions)
A16891, by ABclonal (1 mentions)
A20798, by ABclonal (1 mentions)
As014, by ABclonal (1 mentions)
A19607, by ABclonal (1 mentions)
Ac002, by ABclonal (1 mentions)
Ripa lysis buffer, by Meilun (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
Modified ripa buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
6 protocols using chemiluminescence imaging system
1
Protein Expression Analysis in Cancer Cells
2
Western Blot Analysis of Lung Cancer Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this lung cancer investigation, Western blot analysis was conducted with proteins extracted from human lung adenocarcinoma cell lines (A549) and normal lung fibroblast cells (MRC-5). Protein extraction entailed lysing cells in RIPA buffer supplemented (Beyotime, Shanghai, China) with a PMSF protease inhibitor (CoWin Biosciences, Jiangsu, China) at a 1:100 ratio. Total protein was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Guangzhou, China). After quantification, each protein sample, measuring 10 μg, was loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, proteins were transferred to a 0.45 μm polyvinylidene difluoride membrane. These membranes were initially incubated with primary antibodies overnight at 4 °C, targeting KRT6A (BS, 1:1000), HMMR (BS, 1:1000), FAM83A (BS, 1:1000), and GAPDH (CST, 1:1000) as a standard reference. Protein band visualization was achieved using a chemiluminescence imaging system (Beijing, China) and an ECL chromogenic substrate kit (Thermo Fisher Scientific, Guangzhou, China).
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
The treated cells were harvested and lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM PMSF (Beyotime, Shanghai, China), protease inhibitor cocktail, (Beyotime, Shanghai, China) and phosphatase inhibitors (Beyotime, Shanghai, China). Cytosol and nucleus proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China). The concentrations of protein were determined by the BCA Protein Assay Kit (Thermo Scientific, LOT UA276918, USA). Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (0.45 μm) (Millipore, Ireland, LOT R1BB08547). The membranes were incubated with the following antibodies: β-catenin (1 : 1,000; CST 8480S), Lamin-b1 (1 : 1,000; CST 13435S), and GAPDH (1 : 1,000; CST 5174T). Subsequently, all membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (CST 7074 S) for 2 hr at room temperature. The bands were detected using the Chemiluminescence imaging system (BG-gdsAUTO710Pro, Shanghai) with PierceTM ECL Western Blotting Substrate (Thermo, #32106) and quantified using the Image J software (National Institutes of Health, USA).
4
Protein Expression Analysis in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer (MA0151, meilunbio, Wuhan, China) containing protease inhibitor on ice. Protein extracts (30 µg) were separated by electrophoresis on SDS-polyacrylamide gels, then transferred to PVDF membrane, blocked with milk for 1 h at room temperature, and then incubated with antibodies overnight. Wash three times using TBST. Add secondary antibody (1:5000, AS014, ABclonal, Wuhan, China) and incubate at room temperature for 1 h. Use TBST to wash after imaging on a chemiluminescence imaging system (Thermo Fisher Scientific, Massachusetts, USA). The following antibodies were used: Anti-GAPDH (1:1000, AC002, ABclonal, Wuhan, China), Anti-TGF-β1 (1:1000, A21244, ABclonal, Wuhan, China), Anti-ACE2 (1:1000, AC002, ABclonal, Wuhan, China), Anti-USP4 (1:1000, A20005, ABclonal, Wuhan, China), Anti-COL1A1 (1:1000, A16891, ABclonal, Wuhan, China), Anti-ACTA2 (1:1000, A1011, ABclonal, Wuhan, China), Anti-Vimentin (1:1000, A19607, ABclonal, Wuhan, China), Anti-E-Cadherin (1:1000, A20798, ABclonal, Wuhan, China).
5
Western Blotting Analysis of CAF-1/p150 in HeLa and SiHa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HeLa and SiHa cells were lysed in modified RIPA buffer (Beyotime Institute of Biotechnology), containing Tris-HCl 50 mM, pH: 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, 1 mM NaF) and centrifuged at 13,000 × g for 30 min at 4°C. Protein concentration was determined using a bicinchoninic acid protein assay. Equal amounts of protein (50 µg) were separated via 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore). Membranes were blocked with 5% non-fat milk for 1 h at 37°C and subsequently incubated with CAF-1/p150 antibody (1:5,000) overnight at 4°C. Following the primary incubation, membranes were incubated with anti-rabbit secondary antibody for 1 h at 37°C. Protein bands were detected using the Horseradish Peroxidase Color Development kit (cat. no. p0018s-1; Beyotime Institute of Biotechnology), visualized using a chemiluminescence imaging system (Thermo Fisher Scientific, Inc.) and evaluated using Image Lab software version 6.0 (Bio-Rad Laboratories).
6
Bimolecular Fluorescence Complementation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TaTHI2 CDS was cloned into pCambia1300‐nluc and pCambia1300‐cluc to produce TaTHI2‐nluc and pcLuc‐TaTHI2. TaCPK5 CDS was cloned into pCambia1300‐nluc and pCambia1300‐cluc to produce TaCPK5‐nluc and pcLuc‐TaCPK5. TaCAT1 CDS was cloned into pCambia1300‐nluc to produce TaCAT1‐nluc. CWMV CP was cloned into pCambia1300‐cluc to produce pcLuc‐CP. These plasmids were separately transformed into Agrobacterium strain GV3101. After culturing and induction, Agrobacterium culture carrying pcLuc‐TaTHI2 was mixed in equal proportions with Agrobacterium culture carrying TaCPK5‐nluc. Agrobacterium culture carrying pcLuc‐TaCPK5 was mixed in equal proportions with Agrobacterium culture carrying TaCAT1‐nluc. Agrobacterium culture carrying pcLuc‐CP was mixed in equal proportions with Agrobacterium culture carrying TaTHI2‐nluc. Then, the mixed Agrobacterium cultures infiltrated into N. benthamiana leaves individually. After 72 h, the leaves were incubated with a 0.2 mm luciferin (Thermo Scientific, USA) solution for 5 min and then observed under a chemiluminescence imaging system (Tanon). cLuc‐TaTHI2+nLuc, cLuc‐TaCPK5+nLuc, cLuc‐CP+nLuc, cLuc+TaCPK5‐nLuc, cLuc+TaCAT1‐nLuc and cLuc+TaTHI2‐nLuc were used as negative controls, cLuc‐SAR+sGF‐nLuc were used as positive controls.
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!