For this, 5 × 106 cells in 10 cm dish were lysed by NP40 buffer (Solarbio, N8031) containing 1× cocktail (Roche, 05892970001). Five hundred micrograms of protein was incubated with 5 µg RIgG or HSF1 antibody for overnight with rotating at 4°C. Then, 50 µl agarose beads was added to the mixture for 3 h with rotating at 4°C. The mixture was subjected to Western blot for detection.
Np40 buffer
Manufactured by Solarbio
Sourced in China
NP40 buffer is a non-ionic detergent used for cell lysis and protein extraction. It is a versatile tool for a variety of applications in molecular biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Beyotime (1 mentions)
Protein a g magnetic beads, by Beyotime (1 mentions)
Ptch1, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Chrm1, by Abcam (1 mentions)
Ecl blotting detection kit, by Bio-Rad (1 mentions)
Bicinchoninic acid bca method, by Beyotime (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
4 protocols using np40 buffer
1
HSF1 Protein Interaction Assay
2
Immunoprecipitation of USP21 and p65
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Bladder cancer cells were harvested and lysed using NP-40 buffer (#N8032, Solarbio) containing a complete protease inhibitor cocktail (#P1006, Beyotime Biotechnology) on ice for 30 min. The obtained cell lysate was then centrifuged for 15 min at 12,000 rpm at 4 °C. The lysate supernatant was served as input, and the rest was cocultured overnight with Protein A/G Magnetic Beads (#P2179M, Beyotime Biotechnology) and the anti-USP21 or anti-p65 antibody on a shaking device at 4 °C. The corresponding IgG (Beyotime Biotechnology) was defined as the negative control. Subsequently, the magnetic beads were rinsed using cold NP-40 buffer and boiled for 10 min in loading buffer. After final centrifugation, the supernatant was harvested for western blot analysis with the primary antibodies as displayed.
3
Carbachol and Pirenzepine Modulate Hedgehog Signaling
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PC-3 cells were seeded in 6-well or 12-well plates and left untreated (control) or treated with carbachol (2 μg ml−1) or pirenzepine (110 μg ml−1) for 2, 4, 6, 12, and 24 h. Cells were lysed in NP 40 buffer (Solarbio, Beijing, China) containing protease inhibitors. Total protein was measured using the Bicinchoninic acid (BCA) method (Beyotime). Primary antibodies against SHH (1:2000, Abcam, Cambridge, MA, USA), PTCH1 (1:1000, Abcam), GLI1 (1:1000, CST, Beverly, MA, USA), and CHRM1 (1:1000, Abcam) as well as horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSbio, Beijing, China) were applied. Equal protein sample loading was monitored using an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000, Ray Antibody, Beijing, China). The probed proteins were visualized with an enhanced chemiluminescence (ECL) blotting detection kit (BIO-RAD, Chengdu, China).
4
Western Blot Analysis of Protein Targets
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Western blotting was performed as previously described. Briefly, total protein was extracted using NP40 buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 4 °C for 20 minutes, which was followed by centrifugation at 12,000 ×g at 4 °C for 10 minutes. Proteins were separated via 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Beijing Solarbio Science & Technology Co., Ltd.). Following blocking with 5% bovine serum albumin (BSA) in phosphate-buffered saline with Tween 20 (PBST; 0.1% Tween20) at room temperature for 1 hour, the membranes were incubated with primary antibodies [A20 (from cell signaling technology Co., Ltd, USA, lot #5630), GAPDH (from cell signaling technology Co., Ltd, USA, LOT #5174)], at 4 °C overnight. Subsequently, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (from cell signaling technology Co., Ltd, USA, lot # 7076) at 37 °C for 1 hour. Protein bands were visualized using enhanced chemiluminescence (ECL) reagent. GAPDH was used as the loading control.
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