MLO-Y4 and MC3T3-E1 cells were collected using a cell scraper, treated with Np-40 cell lysis buffer (Beyotime, Shanghai, China), and placed on ice, followed by centrifugation and collection of the supernatant. The protein concentration was detected and was adjusted to 1 μg/μL using Np-40 cell lysis buffer (Beyotime, Shanghai, China). Subsequently, 25 μL of protein G magnetic beads (Thermo, 01108614) were added for pre-cleaning. After incubation with gentle shaking, the magnetic beads were separated using a magnetic rack and the supernatant was collected for protein analysis. Rabbit IgG and anti-Cx43/β-catenin primary antibodies were then added to the protein followed by incubation. After this, 35 μL of magnetic beads were added with gentle shaking. The magnetic beads were collected using a magnetic rack and transferred to a new tube. Bound proteins were eluted with 2× protein-loading buffer after boiling. Cx43 and β-catenin were detected by Western blotting, as previously described.
Np 40 cell lysis buffer
Manufactured by Beyotime
Sourced in China
NP-40 cell lysis buffer is a non-ionic detergent used for the extraction and isolation of proteins from cells and tissues. It functions by disrupting cell membranes, solubilizing membrane-bound proteins, and releasing cellular contents without denaturing the proteins.
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Lab products found in correlation
Anti flag affinity resin a2220, by Merck Group (7 mentions)
Anti flag f1804, by Merck Group (4 mentions)
Anti myc 05 419, by Merck Group (3 mentions)
Ab52644, by Abcam (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Np 40, by Beyotime (2 mentions)
Alexa fluor 546 conjugated anti mouse or anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Mouse anti myc 05 419 and anti flag f1804 mabs, by Merck Group (2 mentions)
Mouse anti flag f1804, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide nem, by Merck Group (1 mentions)
Mg132 s2619, by Selleck Chemicals (1 mentions)
Ab7780, by Abcam (1 mentions)
Mouse anti ha h3663, by Merck Group (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Phosphorylase inhibitor, by Beyotime (1 mentions)
23 protocols using np 40 cell lysis buffer
1
Immunoprecipitation of Cx43 and β-Catenin
2
Antibody-based SADS-CoV Detection Protocol
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Anti-GAPDH mouse monoclonal antibody (mAb), anti-Flag mAb, and anti-HA mAb were obtained from Proteintech (Wuhan, China). Glutathione agarose beads for GST pull-down were purchased from BEAVER (Suzhou, China), and Lipofectamine 3000 was purchased from Life Technologies (Invitrogen, USA). Cell NP-40 lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40] was purchased from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse and anti-rabbit immunoglobulin G (IgG) were purchased from KPL (Milford, MA, USA). Mouse mAb to N of SADS-CoV was produced by our laboratory.
3
Antibody reagents for IBDV research
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Rabbit polyclonal antibody (pAb) against Myc (R1208-1), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; EM1101), and β-actin (EM21002) were purchased from Huaan Biological Technology (Hangzhou, China). Mouse anti-HA (H3663) and anti-Flag (F1804) MAbs were both obtained from Sigma-Aldrich (USA). Anti-Flag affinity resin (A2220) for immunoprecipitation was also purchased from Sigma-Aldrich. Rabbit pAb against ubiquitin (ab7780) were purchased from Abcam (USA). Rabbit anti-K63 (D7A11) and anti-K48 (D9D5) MAbs were obtained from Cell Signaling Technology (USA). Rabbit pAb to VP1 of IBDV, chicken pAb to VP3 of IBDV, and mouse MAb to VP1, VP2, VP3, VP4, and VP5 of IBDV were produced by our laboratory (17 (link), 51 (link), 52 (link)). MG132 (S2619) was purchased from Selleckchem (USA). Cycloheximide (CHX) was obtained from Medchemexpress (HY-12320). N-ethylmaleimide (NEM), a deubiquitination inhibitor, was obtained from Sigma-Aldrich (E3876). Cell NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40) was purchased from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)- or fluorescein isothiocyanate (FITC)-labeled goat anti-mouse and anti-rabbit IgG were purchased from KPL (Milford, MA). FITC-labeled goat anti-chicken antibodies were purchased from Abcam (ab6873). Alexa Fluor 546-conjugated anti-mouse or anti-rabbit IgG were obtained from Invitrogen (USA).
4
Antibody Reagents for Protein Analysis
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Rabbit polyclonal antibody (pAb) against Myc (R1208-1), GFP (SR48-02), FLAG (0912-1), and mouse monoclonal antibody (mAb) against β-actin (M1210-2), GST (M0807-1) were purchased from Huaan Biological Technology (Hangzhou, China). Mouse anti-Myc (05-419) and anti-FLAG (F1804) mAbs were both obtained from Sigma-Aldrich (United States). Anti-FLAG affinity resin (A2220) for immunoprecipitation was also purchased from Sigma-Aldrich. Mouse monoclonal antibody (mAb) against GFP (B-2, sc-9996) was purchased from Santa Cruz Biotechnology. Glutathione agarose beads for GST pull-down were purchased from Thermo Scientific (United States). Mouse mAb to Cap of PCV2 was produced by our laboratory (Zhou et al., 2005 (link)). Cell NP-40 lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40] was purchased from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse and anti-rabbit IgG were purchased from KPL (Milford, MA, United States).
5
Immunoprecipitation of GFP- and His-tagged Proteins
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The immunoprecipitation analysis was performed based on these protocols (47 (link)). The hepatocytes were lysed in the NP40 cell lysis buffer (p0013F, Beyotime, Shanghai, China) with phosphorylase inhibitor (P1082, Beyotime, Shanghai, China). Then, the anti-GFP tag or anti-His tag was added to the cell lysate overnight at 4 °C and the protein A/G beads (P2012, Beyotime, Shanghai, China) were added. Finally, the immunocomplexes were washed by NP40 buffer. The western blot was used to measure the protein expression.
6
Immunoprecipitation of FLIP Ubiquitination
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Immunoprecipitation was performed as previously described [19 (link)]. Briefly, MSCs were lysed with NP-40 cell lysis buffer (#P0013F, Beyotime) supplemented with a 1× protease phosphatase inhibitor cocktail mixture, 10 mM N-ethylmaleimide (NEM) (# HY-D0843, MCE), and 1 mM EDTA. In total, 10% of the lysates was kept as input, and the remaining samples were used for immunoprecipitation. Rabbit monoclonal anti-FLIP antibodies (1 : 100, #56343, CST) or normal rabbit IgG was incubated with magnetic protein A beads (#1614013, Bio-Rad) for 1 h at room temperature. The supernatant was discarded, and the bead–antibody complexes were washed with lysis buffer 3 times. Then, the bead–antibody complexes were incubated overnight at 4°C with the cell lysates mentioned above. The bead–antibody–protein immunocomplexes were washed 3 times with lysis buffer and then boiled in loading buffer with SDS at 98°C for 5 min to elute the proteins from the beads. Immunoblotting was performed to analyze the ubiquitin levels.
7
Co-Immunoprecipitation in 293T Cells
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293T cells were transfected with the respective plasmids using Polyjet (SL100688, SignaGen Laboratories), according to the manufacturer’s instructions. After 48 h, the transfected 293T cells were washed three times with ice-cold PBS and then lysed in NP-40 cell lysis buffer (P0013F, Beyotime Biotech Inc). After centrifugation, 40 μl of the supernatant was collected as the input fraction. The remaining supernatant was collected as the Co-IP fraction, and it was incubated with protein A resin at 4 °C for 6-8 h. Then the protein A affinity matrix beads were collected by centrifugation at 1000g for 5 min at 4 °C and washed five times with ice-cold PBS. The immunoprecipitated proteins were separated by SDS-PAGE and detected by Western blotting. All the experiments were performed with three biological replicates.
8
Cytokine Quantification in Intestinal Tissue
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The intestinal tissue was washed with PBS twice, and grounded with liquid nitrogen till no granule was observed, followed by 30-min lysis using 1 ml NP-40 cell lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China) on ice to collect supernatant protein liquid. Finally, ELISA kits (Nanjing Jiancheng Institute of Biology, Nanjing, China) were employed to determine cytokine levels following instructions.
9
Antibody sources for immunodetection
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Mouse monoclonal antibodies (mAbs) against GST (M0807-1), histone H3 (R1105-1), and β-actin (M1210-2), as well as rabbit polyclonal antibodies (pAbs) against Myc (R1208-1), Flag (0912-1), β-tubulin (0807-2), and GFP (SR48-02) were purchased from Huaan Biological Technology (Hangzhou, China). Mouse anti-Flag (F1804) and anti-Myc (05–419) mAbs were obtained from Sigma-Aldrich (St. Louis, MO, United States). Rabbit mAb against DDX21 (ab182156) was obtained from Abcam (Cambridge, MA). Anti-Flag affinity resin (A2220) for immunoprecipitation was purchased from Sigma-Aldrich. NP-40 cell lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40) was obtained from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse and anti-rabbit IgG or fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG were purchased from KPL (Milford, MA, United States). Alexa Fluor 546-conjugated donkey anti-rabbit IgG was obtained from Invitrogen (United States).
10
Quantitative Analysis of Caveolin-1 Expression
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Western blot analysis was performed to determine protein expression of CAV1. Cell lysates were prepared by using NP-40 cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the protein concentration in the supernatants was determined using Bradford protein dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 30 mg proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membranes were blocked with 5% fat-free dry milk in PBS for 30 min at room temperature and then incubated with primary anti-CAV1 (1:1,000, cat. no. ab2910; Abcam, Cambridge, MA, USA) and anti-GAPDH (1:1,000, cat. no. ab9485; Abcam) antibodies at 4°C overnight. Subsequently, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies [goat anti-rabbit IgG H&L (HRP); 1:3,000, cat. no. ab6721; Abcam] for 2 h at room temperature. CAV1 and GAPDH proteins were visualized with enhanced chemiluminescence detection reagent (Pierce Biotechnology; Thermo Fisher Scientific, Inc.).
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