The cells were harvested, lysed using ice-cold NP-40 lysis buffer (Cell Signaling Technology), centrifuged and the supernatant collected. Equal amounts of the supernatant protein (50 μg) were loaded onto an SDS-PAGE gel. The samples were electrophoresed and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). WB analysis of cell lysates [28 (link),29 (link)] was carried out using the following primary antibodies: SOCS5 (ab244384, 1:2000), Bcl-2 (ab182858, 1;1000), p62 (ab280086, 1:2000), LC3B (ab221794, 1:2000), Beclin 1 (ab207612, 1:2000), ATG7 (ab52472, 1:2000), Bcl-2 (phospho S70, ab218123, 1:2000), cleaved Caspase-3 (ab32042, 1:3000), cleaved Caspase-9 (ab2324, 1:3000), and GAPDH (ab9485, 1:1000), which were purchased from Abcam. After the membranes were washed three times for 10 min with Tween-Tris-buffered saline buffer, they were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 120 min. The positive signal for the target protein was analyzed using a Tanon 2500 image analyzer (Shanghai, China). Densitometry of specific blotted bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ ), and the intensity values were normalized against the GAPDH loading control.
Np 40 lysis buffer
Manufactured by Cell Signaling Technology
NP-40 lysis buffer is a non-ionic detergent solution designed for the extraction and solubilization of proteins from cells and tissues. It is commonly used to prepare cell lysates for various downstream applications such as protein analysis, immunoprecipitation, and enzyme assays.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Ab52472, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab221794, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Anti ubiquitin ab, by Cell Signaling Technology (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
4 protocols using np 40 lysis buffer
1
Western Blot Analysis of Autophagy Proteins
2
SPOP and CDK1 Interactome Analysis
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Briefly, 293T cells were transfected with SPOP and CDK1 or empty vector (control) expression vectors. Forty hours after the transfection, 500 nmol/L (final concentration) PS-341 was added to incubation for another 8 hours. The cells were collected, washed by PBS, and lysed in NP-40 lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (MilliporeSigma). Cell lysates were incubated with protein-specific antibodies and the immunocomplexes were collected by incubating the lysate-antibody mix with protein G Dynabeads at 4°C on a rotator. After extensive washing with NP-40 lysis buffer, the immunocomplexes were eluted from the beads by 1xSDS loading buffer and analyzed by immunoblotting.
3
SPOP and CDK1 Regulation in 293T Cells
4
Endogenous Wee1/p27/p21 Ubiquitination Assay
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To detect endogenous Wee1/p27/p21 ubiquitination, 1 × 106 Miapaca-2 and Capan-1 cells were treated with pevonedistat vs. DMSO for 24 h and further treated with MG-132 for more than 24 h. After that, cells were harvested and lysed in NP-40 lysis buffer (Beyotime, catalog: P0013F) under denaturing conditions. After centrifugation at 4°C, cell lysates were incubated with 5 μl of anti-Wee1/p27/p21 Ab or rabbit IgG control antibody at 4°C overnight. Ten microliters of Protein A+G agarose (Santa Cruz Biotechnology) was then added to the cell supernatant for incubation at 4°C for 1 h. Following washing 6 times with NP-40 lysis buffer, the beads were subjected to IB with anti-ubiquitin Ab (Cell Signaling).
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