The level of total protein in the supernatant of the NSCLC cell lysis was determined by the BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, China). The samples were boiled for 10 min at 95°C. An aliquot of 30 mg of protein was separated using 10% SDS–PAGE gel, followed by PVDF membrane transfer, blocking for 1 h in 5% skim milk, and incubation with separate antibodies (ABCAm, Inc., USA)—1:1000 diluted antibody Ab127548 (anti-RNF180), 1:500 diluted antibody Ab39688 (anti-c-Myc), 1:5000 diluted antibody Ab227198 (anti-HK-2), 1:1000 diluted Ab101562 antibody (anti-LDHA), and 1:2000 diluted anti-GAPDH antibody (#5174, Cell Signaling Technology)—overnight at 4°C. Thereafter, the membranes were incubated with horseradish peroxidase secondary antibodies (A0208 (1:200), A0181(1:200), and A0216(1:200); Beyotime Biotechnology) at room temperature for 1 h. The ECL plus substrate (GE Healthcare, USA) with the LAS-400 Image Analyzer (FujiFilm Medical Systems, USA) was used for the detection of the horseradish peroxidase signal.
Las 400 image analyzer
Manufactured by Fujifilm
Sourced in United States
The LAS-400 is an image analyzer manufactured by Fujifilm. It is designed to capture and analyze images. The device features high-resolution image capture capabilities and advanced image processing functions.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab127548, by Cell Signaling Technology (2 mentions)
Ab227198, by Cell Signaling Technology (2 mentions)
A0216, by Beyotime (2 mentions)
Ecl plus substrate, by GE Healthcare (2 mentions)
A0208, by Beyotime (2 mentions)
Ab101562, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Thermo Fisher Scientific (1 mentions)
A0181, by Beyotime (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab202445, by Abcam (1 mentions)
Ab63399, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
Ab14824, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab127574, by Abcam (1 mentions)
Ecl kit, by Abcam (1 mentions)
5 protocols using las 400 image analyzer
1
Quantification of Protein Expression in NSCLC
2
Protein Expression Analysis by Western Blot
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Total protein concentration was determined by BCA protein assay kit according to the manufacturer's instructions (Thermo, MA, USA). Samples were heated at 95℃ for 10 min, and 30 mg of them was separated by 10% SDS-PAGE. Then, samples were transferred to PVDF membranes and blocked in 5% skim milk for 1 h at room temperature(RT). The membrane was incubated with the primary antibody USP49 (1:1000, ab127574, Abcam, UK), PPM1A (1:1000, ab14824, Abcam), vimentin (1:500, ab8978, Abcam), α-SMA (1:1000, ab124964, Abcam), E-cadherin (1:500, ab1416, Abcam), Smad2/3 (1:1000, ab202445, Abcam), p-Smad2/3 (1:500, ab63399, Abcam), TGF-β1 (1:1000, ab179695, Abcam), and GAPDH (1:2000, #5174, CST, MA, USA) at 4℃ overnight. Then, the membrane was incubated with secondary antibodies (A0208, A0181, and A0216, GE Healthcare/Amersham Biosciences, Piscataway, NJ, China) at RT for 1h. LAS-400 image analyzer (FujiFilm Medical Systems, CT, USA) was used to detect the HRP (GE Healthcare/Amersham Biosciences) signal.
3
Protein Analysis in Colorectal Cancer
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Total protein extracts were prepared from CRC cells or tumor tissues using RIPA buffer and were quantified by the BCA protein assay kit (Thermofisher, Bridgewater Township, NJ, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated with anti-CPNE1, anti-GLUT1, anti-HK2, anti-cleaved Caspase 3 (Abcam, Cambridge, MA), anti-AKT, anti-p-AKT, or anti-GAPDH antibodies (Santa Cruz, Santa Cruz, CA) at 4°C overnight. Bands were visualized using an ECL kit (BioVision, Exton, PA) with an LAS-400 image analyzer (FujiFilm Medical Systems, Stamford, CT).
4
Protein Expression Analysis in NSCLC Cells
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The total protein level in the supernatant of NSCLC cell lysis was determined by BCA (bicinchoninic acid) protein assay kit (Thermo Fisher Scienti c, China). The samples were boiled for 10 min at a temperature of 95°C. An aliquot of 30 mg of protein was separated by SDS-PAGE (10% gel) followed by PVDF membranes transfer, 1 h blocking in 5% skim milk, and incubation with separate antibodies (Abcam, Inc., USA) of 1:1000 diluted antibody Ab127548 (anti-RNF180), 1:500 diluted antibody Ab39688 (anti-C-myc), 1:5000 diluted antibody Ab227198 (anti-HK-2), 1:1000 diluted Ab101562 antibody (anti-LDHA), and 1:2000 diluted antibody against GAPDH (#5174, Cell Signaling Technology) overnight at 4°C. Then the membranes were incubated with horseradish peroxidase secondary antibodies (A0208, A0181 and A0216, Beyotime Biotechnology) at room temperature for 1 h. The ECL plus substrate (GE Healthcare, USA) with LAS-400 image analyzer (FujiFilm Medical Systems, USA) was used for the detection of horseradish peroxidase signal.
5
Protein Fractionation and Western Blot Analysis
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Lysates were made using a radio-immunoprecipitation assay buffer with proteinase inhibitors (Sigma, Shanghai, China). According to the manufacturer's protocol, the cytosolic or nuclear parts were made using a nuclear/cytosol fractionation kit (Biovison, Milpitas, CA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Bio-Rad, Philadelphia, PA). Membranes were blocked with 3% BSA, incubated overnight at 4°C with primary antibodies (Table S1 ), washed, and incubated with HRP-conjugated rabbit secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 hour. Protein bands were detected using enhanced chemiluminescence (Amersham Biosciences, Marlborough, MA) with a LAS400 image analyzer (FujiFilm, Stamford, CT).
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