Cells in control, non-transfected ATF6 (151-366aa) and ATF6 (1-366aa) groups were harvested and washed twice with PBS, and protein lysed in ice-cold radioimmunoprecipitation assay buffer (Whiga Technology Co., Ltd., Guangdong, China) with freshly added 0.01% protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich Co., LLC, Darmstadt, Germany). The cells were incubated on ice for 30 min and then centrifuged at 10,000 × g for 5 min at 4°C. The supernatant (20-30 µg of protein) was collected, resolved by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc.), and subsequently transferred to a nitrocellulose membrane via western blotting (Millipore, Shanghai, China). Then the expression of protein cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, cleaved-caspase-4, cleaved-caspase-12, CHOP, cyt c, Bax, Bcl-2, pro-caspase-8, cleaved-caspase-8, Fas, phosphorylated-JNK, JNK and NF-κB were detected. Protein loading was estimated using mouse anti-β-actin monoclonal antibody (Beijing Solarbio Technology Co., Ltd.). Blottings were visualized using enhanced chemiluminescence (Thermo Fisher Scientific Inc.).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Manufactured by Bio-Rad
Sourced in United States
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used to separate and analyze proteins based on their molecular weight. It utilizes an electric current to move charged proteins through a polyacrylamide gel, allowing for the separation and visualization of individual protein bands.
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Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin monoclonal antibody, by Solarbio (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl chemiluminescence staining assay kit, by Bio-Rad (1 mentions)
Cd81 b 11, by Santa Cruz Biotechnology (1 mentions)
Cd63 h5c6, by BD (1 mentions)
Tsg 101 4a10, by Abcam (1 mentions)
Gm130, by Abcam (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl xl, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Cleaved caspase 9, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Dynabeads m 280, by Thermo Fisher Scientific (1 mentions)
8 protocols using sodium dodecyl sulphate polyacrylamide gel electrophoresis
1
Apoptosis Pathway Protein Analysis
2
Protein Characterization of Small and Large Extracellular Vesicles
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The pellets of sEVs and lEVs were lysed with RIPA buffer (Pierce, Rockford, IL, USA) on ice for 40 min. Insoluble material was pelleted by centrifugation for 15 min at 11,000 × g at 4°C. Supernatants were transferred to a new tube, and the protein concentrations were quantified with a BCA Protein Assay kit (Beyotime, Shanghai, China) according to the manufacture’s protocols. Next, 20 μg of protein was loaded into gels for separation by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) after which the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk powder, primary antibodies were added, and the membrane was incubated at 4°C overnight. Primary antibodies used were CD9 (C4, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500), CD81 (B11, Santa Cruz; 1:1000), CD63 (H5C6, BD Biosciences, Franklin Lakes, NJ, USA; 1:1000), Tsg101 (4A10, Abcam, Cambridge, UK; 1:2000), GM130 (4A10, Abcam, Cambridge, UK; 1:500) and Calnexin (Roteintech Group, INC; 1:1000). After washing with PBS, the membrane was further incubated with the secondary antibody for 1 h at room temperature. Proteins were visualized using an ECL chemiluminescence staining assay kit (Bio-Rad) and the density of each protein band was quantified.
3
Western Blot Analysis of Apoptosis Markers
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RIPA lysis buffer (Solarbio, Beijing, China) was used to lyse transfected cells, while phenylmethylsulphonyl chloride was used to prevent degradation of the protein. Bicinchoninic acid (BCA) assay (Thermo Scientific, USA) was used for quantification of the protein. Then, each sample was separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (BioRad, Berkeley, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). PVDF membranes were blocked by 5% non‐fat milk for two hours at room temperature and then incubated with primary antibodies overnight at 4°C. Primary antibodies were as follows: IMUP, BCL‐2, BCL‐XL (Abcam, Cambridge, MA, USA); YAP1, TAZ, BAX, Cleaved‐Caspase9 and β‐Actin (Proteintech, Wuhan, China). After being washed by TBST (Tris‐buffered saline/ 0.1% Tween 20), the membrane was incubated with anti‐rabbit IgG or anti‐mouse IgG (1:5000. Abcam, Cambridge, MA, USA) for two hours at room temperature. Finally, the chemiluminescence kit (Thermo Scientific, USA) was used to visualize the blots, and images of the results were analysed with ImageJ software (NIH).
4
Western Blot Analysis of EMT Markers
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Total protein lysates were separated by 10% of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (Bio‐Rad, Berkeley, CA, USA) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blotted with 5% non‐fat milk for in TBST 2 hours at room temperature, then, washed three times by TBST and probed with rabbit anti‐N‐cadherin (1:2000 dilution), vimentin (1:2000 dilution), UPP1 (1:1000 dilution) and mouse‐β‐actin (1:2000 dilution) according to the company's protocol overnight at 4˚C. The membranes were then incubated with the antimouse IgG or anti‐rabbit IgG as a secondary antibody for 1.5 hours at room temperature. Each antibody was acquired from Abcam (Cambridge, MA, USA).
5
RNA Interactome Identification from Cell Lysates
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Purified RNA was dephosphorylated using calf intestine phosphatase (Roche). For 5′ end biotinylation, 600 pmol RNA were incubated with 0.2 mM γ-S-ATP (Biomol) and T4 polynucleotide kinase (New England Biolabs) for 30 min at 37°C. Biotin-long-arm maleimide (Vector Laboratories) was added and incubated for 30 min at 65°C. Unincorporated label was depleted by LiCl precipitation. Biotinylated RNA (200 pmol) was conjugated to Dynabeads M-280 (Invitrogen) in incubation buffer (10 mM Tris–HCl pH 7.4, 150 mM KCl, 0.5 mM DTT, 0.05% NP40, 100 U/ml RNasin) for 2 h at 4°C with continuous rotation. Whole cell protein lysate (1 mg) of HEK293 cells together with 200 μg yeast tRNA (Sigma-Aldrich) and 5 mg Heparin (Sigma-Aldrich) was added to the beads and incubated for 1 h at 4°C followed by 15 min at room temperature with continuous rotation. Beads were washed five times with incubation buffer, resuspended in 30 μl protein loading dye and boiled at 95°C for 10 min. Eluted proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (BioRad).
6
Quantitative Analysis of Cellular Signaling
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to instruction. The purity of RNA was tested by measuring the absorbance (NanoDrop ND‐1000, Thermo Fisher Scientific) and reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara). The expressions of genes were measured with Bio‐Rad iQ5 real‐time PCR detection system, with β‐actin for normalization. The primers for target genes were listed in Table 1 .
Protein was extracted by whole cell lysis assay (KeyGEN), and the concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal amount of protein samples was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio‐Rad Laboratories) and transferred onto a polyvinylidene fluoride membrane (Millipore). Following incubation in specific antibodies, we probed proteins by enhanced chemiluminescence kit (Bio‐Rad Laboratories). Bands were quantified by ImageJ software. Specific antibodies included CREB (Assay biotechnology, 1:1000), pCREB (Assay biotechnology, 1:1000), CDKN2A/p16INK4a (Abcam, 1:1000), pSMAD3 (Abcam, 1:1000) and Tubulin α (Signalway Antibody, 1:2000).
Protein was extracted by whole cell lysis assay (KeyGEN), and the concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal amount of protein samples was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio‐Rad Laboratories) and transferred onto a polyvinylidene fluoride membrane (Millipore). Following incubation in specific antibodies, we probed proteins by enhanced chemiluminescence kit (Bio‐Rad Laboratories). Bands were quantified by ImageJ software. Specific antibodies included CREB (Assay biotechnology, 1:1000), pCREB (Assay biotechnology, 1:1000), CDKN2A/p16INK4a (Abcam, 1:1000), pSMAD3 (Abcam, 1:1000) and Tubulin α (Signalway Antibody, 1:2000).
7
SDS-PAGE Protein Analysis Protocol
8
Cytotoxicity Assay for Cancer Cell Lines
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Dabrafenib (GSK2118436A) and LEE011 (Active Biochem, Hong Kong) were dissolved in DMSO (Sigma-Aldrich) at a final concentration of 1.92 mM and 16 mM, respectively. Drugs were stored as stock solutions at –80°C and diluted in CM just before use.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich, dissolved at a concentration of 5 mg/ml in GIBCO™ Phosphate-Buffered Saline (PBS) (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and stored at 4°C.
Mouse monoclonal antibody (mAb) against PTTG1 (DCS-280) and rabbit polyclonal antibody against human β-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA).
Reagents for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis were all purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich, dissolved at a concentration of 5 mg/ml in GIBCO™ Phosphate-Buffered Saline (PBS) (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and stored at 4°C.
Mouse monoclonal antibody (mAb) against PTTG1 (DCS-280) and rabbit polyclonal antibody against human β-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA).
Reagents for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis were all purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).
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