Pancreatic cancer cell lines were washed and resuspended with PBS twice. Total protein was isolated and quantified using RIPA extraction buffer and the BCA kit (Pierce). The protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with buffer containing 5% non-fat milk and incubated with TLE2 antibody (rabbit polyclonal anti-human, Abcam, USA; 1:200, No. ab206147). Anti-rabbit IgG was used as the secondary antibody (Santa Cruz Biotechnology, No. sc-2357). The blots were visualized with an ELC kit (Millipore, USA).
Elc kit
Manufactured by Merck Group
Sourced in United States
The ELC kit is a laboratory equipment product that provides a core function. It is designed for specific tasks within a laboratory setting. The details of its intended use and applications are not included in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Mammalian protein extraction buffer, by Merck Group (2 mentions)
Quantity 1 version 4, by Bio-Rad (2 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Amersharm, by GE Healthcare (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
Anti fas, by Cell Signaling Technology (1 mentions)
Anti srebp1, by Abcam (1 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Nf κb pathway sampler kit, by Cell Signaling Technology (1 mentions)
β actin 13e5 rabbit mab, by Cell Signaling Technology (1 mentions)
Parp 46d11 rabbit mab, by Cell Signaling Technology (1 mentions)
Ampkα d63g4 rabbit mab, by Cell Signaling Technology (1 mentions)
Bax rabbit mab, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 asp175 5a1e rabbit mab, by Cell Signaling Technology (1 mentions)
Caspase 3 rabbit mab, by Cell Signaling Technology (1 mentions)
4 protocols using elc kit
1
Western Blot Analysis of TLE2 in Pancreatic Cancer
2
Protein Expression Analysis in Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liver tissue and cell lysates were homogenized in mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO. USA) in the presence of protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (Sigma, PMSF). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The protein contents of the supernatants were determined using protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA) based on the manufacturer’s instructions. The same concentration of protein was separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Amersharm, GE Healthcare Life Science, Germany). The antibodies used for western blotting included anti-SREBP-1 (1:1000; Abcam, Cambridge, UK), anti-ACC (1:1000; Cell Signaling Technology), anti-FAS (1:1000; Cell Signaling Technology), anti-AMPKα (1:1000; Cell Signaling Technology), anti-phospho-AMPKα (Thr172) (1:1000; Cell Signaling Technology), and anti-β-actin (1:2500; Abcam). The protein bands were visualized following an enhanced chemiluminescence method using an ELC kit (Millipore, USA). The bands were quantified using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories).
3
Isolation and Analysis of Nuclear Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear protein was isolated from INS-1 cells using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, MA, USA) and evaluated using the NF-κB pathway sampler kit (Cell Signaling Technology, Danvers, MA, USA).
Western blotting was conducted as described previously [11 (link)]. Briefly, 10–50 mg of proteins was separated by 10% SDS–PAGE, transferred to nitrocellulose blotting membranes, and incubated with primary antibodies. After washing the membranes with TBST, they were incubated with secondary antibodies. Protein bands were detected using an ELC kit (Millipore, Burlington, MA, USA). Bands were normalized to β-actin and quantified using the Quantity 1 version 4.6.7 software (Bio-Rad, Hercules, CA, USA).
Western blotting was conducted as described previously [11 (link)]. Briefly, 10–50 mg of proteins was separated by 10% SDS–PAGE, transferred to nitrocellulose blotting membranes, and incubated with primary antibodies. After washing the membranes with TBST, they were incubated with secondary antibodies. Protein bands were detected using an ELC kit (Millipore, Burlington, MA, USA). Bands were normalized to β-actin and quantified using the Quantity 1 version 4.6.7 software (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Protein Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell lysates were homogenized in mammalian protein extraction buffer (Sigma). The resulting lysates were centrifuged at 12,000 rpm, and 4°C for 15 min, and the protein contents were measured using a protein-assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 10–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, followed by transfer onto nitrocellulose blotting membranes (Amersham, GE Healthcare Life Science, Germany). The antibodies used for immunoblotting are as follows: Bax rabbit mAb (1:1,000; Cell Signaling Technology), Bcl2 (D17C4) rabbit mAb (1:1,000; Cell Signaling Technology), inducible nitric oxide synthase (iNOS; NOS2) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AMPKα (D63G4) rabbit mAb (1:1,000; Cell Signaling Technology), Phospho-AMPKα (Thr172) (D4D6D) rabbit mAb (1:1,000; Cell Signaling Technology), caspase-3 rabbit mAb (1:1,000; Cell Signaling Technology), cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (1:1,000; Cell Signaling Technology), PARP (46D11) rabbit mAb (1:1,000; Cell Signaling Technology), and β-actin (13E5) rabbit mAb (1:2,500; Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence method with an ELC kit (Millipore, Billerica, MA, USA) and were quantified using Quantity 1 v.4.6.7 (Bio-Rad).
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!