Total protein, membrane protein, cytoplasmic protein, and nucleoprotein were extracted from RA FLS and HEK293T cells by ProteoPrep® Total Extraction Sample Kit (Sigma-Aldrich), ProteoPrep® membrane Extraction Kit (Sigma-Aldrich), and NE-PER™Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), respectively. Protein in culture medium (100 ml) was pretreated by freeze-drying method and then dissolved in an appropriate amount of double distilled water. After quantification with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), 25 μg protein samples were separated by SDS-PAGE and then electrotransfered onto polyvinylidene difluoride membranes. After blocked in 5% skim milk for 2 h, the membranes were incubated with antibodies of KIAA1199 (#ab98947, Abcam), HYAL2 (#ab126099, Abcam), CD44 (#ab157107, Abcam), AXNA1 (#ab88865, Abcam), PI3K (#4249, CST), total-Akt (#2920, CST), p-Akt (Ser473) (#4060, CST), total-STAT3 (#4904, CST), p-STAT3 (Tyr705) (#9131, CST), p-P65 (Ser536) (#3036, CST), and then with matching HRP-conjugated secondary antibodies. Protein bands were visualized using ECL substrate (#35055, Thermo Fisher Scientific). β-Actin (#AP0060, Bioworld) and Lamin B1 (#12987-1-AP, Proteintech) were used as the internal reference of cytoplasmic protein and nucleoprotein, respectively. The intensity of protein bands were analyzed by Image J (v1.8.0).
Proteoprep total extraction sample kit
Manufactured by Merck Group
Sourced in United States, China
The ProteoPrep® Total Extraction Sample Kit is a laboratory equipment product designed for the extraction of proteins from various sample types. It provides a complete set of reagents and tools necessary for the efficient isolation and purification of proteins from a wide range of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ab133470, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl detection kit, by Cytiva (2 mentions)
Hrp labeled goat anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Lamin b1, by Proteintech (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Proteoprep membrane extraction kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Gapdh, by Beyotime (1 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (1 mentions)
28 protocols using proteoprep total extraction sample kit
1
Protein Extraction and Western Blot Analysis
2
Proteomic Analysis of hPDLSCs and Exosomes
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RIPA buffer (Beyotime, China) and ProteoPrep® Total Extraction Sample Kit (Sigma-Aldrich, St. Louis, MO, USA) were used to lyse hPDLSCs (osteogenesis induction and exosome treatment at d 7 and 21) and exosomes, respectively, to obtain total protein. The concentration of hPDLSCs and total exosomal proteins was measured using BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were added on SDS-PAGE and electrophoresis was performed, followed by transfer to PVDF membrane. The membrane was blocked with 5% BSA for 2 h at room temperature and the exosome-loaded membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies CD9 (1:1000; ab236630), TSG101 (1:3000; ab125011) and ALIX (1:1000; ab275377). The hPDLSCs samples were incubated overnight with rabbit polyclonal antibodies to ALP (1:1000; ab229126) and OCN (1:2000; ab93876). The membrane was washed 3 times with PBST and incubated with Goat Anti-Rabbit IgG H&L (HRP) (1:5000; abcam; ab205718) for 1 h at 37 °C. β-actin was used as an internal reference. The ECL kit (Beyotime) was used for development and the bands on the membrane were scanned and imaged by E-Gel Imager gel imaging system (Thermo Fisher Scientific). All the above antibodies were purchased from Abcam plc (Cambridge, MA, USA). The results were quantified using Image J software.
3
Protein Expression Analysis in Lung Tissues
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Total protein was isolated from the excised lung tissues by the ProteoPrep total extraction sample kit (Sigma-Aldrich China LLC). The protein samples were transferred to a polyvinylidene fluoride membrane by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein expressions of caspase-3, caspase-9, Bcl-2, Bax, survivin, α-SMA, fibronectin, laminin, collagen I, JAK2, and STAT3 were detected using a standard procedure. The dilution concentration of antibodies used was that recommended by the manufacturer. Protein density was standardized as β-actin, and phosphorylated protein density as total protein.
4
Subcellular Localization and Transcriptional Activation of JcMADS40 in Arabidopsis
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The amplified coding region of JcMADS40 without the termination codon was inserted into this vector (pSAT6-eYFP-N1) to generate 35S::JcMADS40-YFP. The 35S::JcMADS40-YFP fusion expression construct and the 35S::YFP empty vector were transferred into Arabidopsis protoplasts using the polyethylene glycol-mediated method. Subcellular localization of the control YFP and JcMADS40-YFP fusion proteins was observed under a confocal laser scanning microscope. Arabidopsis protoplasts were obtained following Tang [5 (link)].
For the transactivation assay of JcMADS40, the full-length JcMADS40 gene was fused to the pBD vector to generate the construct pBD-JcMADS40. This construct and the p5 × GAL-Reporter vector were introduced into Arabidopsis protoplasts. A ProteoPrep® Total Extraction Sample Kit (Sigma) was employed to isolate total protein of Arabidopsis protoplasts according to the operating instructions of the kit, then the fluorescence activity of proteins was analyzed using the enzyme-labeled instrument. The LUC/REN ratio was used to measure the transcriptional activation of JcMADS40.
For the transactivation assay of JcMADS40, the full-length JcMADS40 gene was fused to the pBD vector to generate the construct pBD-JcMADS40. This construct and the p5 × GAL-Reporter vector were introduced into Arabidopsis protoplasts. A ProteoPrep® Total Extraction Sample Kit (Sigma) was employed to isolate total protein of Arabidopsis protoplasts according to the operating instructions of the kit, then the fluorescence activity of proteins was analyzed using the enzyme-labeled instrument. The LUC/REN ratio was used to measure the transcriptional activation of JcMADS40.
5
Protein Extraction and Western Blotting
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Total protein was extracted from cells using a ProteoPrep® Total Extraction Sample kit (Sigma-Aldrich Co., St Louis, MO, USA). The resulting cell lysis was centrifuged 12,000× g at 4°C for 10 min and supernatant proteins were separated by 10%–15% SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Protein expression was analyzed by Western blot using various antibodies at the following dilutions: TP53 antibody (ab65021; 1:600), caspase-9 antibody (ab32539; 1:800) and tubulin antibody (AmyJet Scientific Co. Ltd, Hubei, China; 1:2,000).
6
Protein Extraction and Western Blot Analysis
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The proteins in cells were isolated by using the ProteoPrep® total extraction sample kit (Sigma, USA). After centrifuging the cells at a speed of 12,000 g for ten minutes at 4°C, the supernatant was subjected to SDS gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Shanghai, China) and blocked for 1 h at room temperature. The membranes were incubated with antibodies against Wnt5a (#2392, CST, USA), Wnt8b (bs-6245R, Invitrogen, USA), and GAPDH (AG109, Beyotime, China). Subsequently, the membranes were incubated with an anti-mouse IgG HRP-linked antibody (#7076, CST, USA), and the bands were developed by using the ECL detection reagent (Sigma, USA).
7
Transcriptional Activation Assay of JcHDZ16
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The full-length cDNA of JcHDZ16 amplified by PCR using specific primers was inserted into the Kpn I and Xba I sites to creat a fusion construct of pBD-JcHDZ16. The pBD-JcHDZ16 plasmid and p5 × GAL-Reporter plasmid were transformed into Arabidopsis protoplasts. An empty pBD vector was used as a negative control. Total protein from Arabidopsis protoplasts was extracted using ProteoPrep® Total Extraction Sample Kit (Sigma) based on the manufacturer’s instructions, and then the enzyme-labeled instrument (TECAN LAI-2000) was used to analyze the fluorescent activity of proteins. Transcriptional activation was analyzed according to the ratio of LUC/REN.
8
Extraction and Analysis of Total Cellular Proteins
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Total protein was extracted from cells using the ProteoPrep® Total Extraction Sample Kit (PROTTOT; Sigma-Aldrich, USA). Samples were heated to denature proteins and stored at −20°C. Twenty microliters of each protein sample were taken for the immunoblotting experiment. Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with non-fat milk for 3 h. The primary antibodies at 1:1,000 dilution (CYP1A2 antibody: ab151728, Abcam, UK; GAPDH antibody: ab8245, Abcam, UK) were, respectively, added to the membrane and incubated overnight. After washing with TBST, ALP-conjugated secondary antibody (1:8,000, ab133470; Abcam, UK) was added to the membrane and incubated for an hour. Finally, the membrane was washed with TBST, and Novex AP chemiluminescent substrate (BioMag, Spain) was added to detect the signal. The gray scale values of the bands were analyzed.
9
Western Blot Analysis of FOXQ1 Protein
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The isolation of total protein was carried out with the ProteoPrep® Total Extraction Sample Kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). The concentration of total protein was quantified via the Bicinchoninic Acid Protein Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein were loaded and separated by SDS-PAGE in a 10% gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Prior to incubation with primary antibodies overnight at 4°C, the membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% of Tween 20 (TBST) at room temperature for 2 hrs. After three washes with TBST, a goat anti-rabbit IgG antibody (1:5,000 dilution; horseradish peroxidase-conjugated secondary antibody; cat. # ab6721; Abcam, Cambridge, UK) was incubated with the membranes at room temperature for 1 hr. Finally, the bands were detected using the Enhanced Chemiluminescence Detection Reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Rabbit anti-human FOXQ1 (ab51340) and anti-human GAPDH (ab181602) antibodies were purchased from Abcam and employed at 1:1,000 dilution.
10
Overexpression of PDX1 in SGC7901 Cells
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The vector of pcDNA3.1(+)-PDX1 was established according to the methods previously published[15 ]. The SGC7901 cells were cultured in 6-well plates and transfected with pcDNA3.1(+)-PDX1 vector (SGC-PDX1) or pcDNA3.1(+) control vector (SGC-pcDNA) using liposome transfection reagent (Lipofectamine™ 2000 Transfection Reagent, ThermoFisher Scientific, United States). After six hours, fresh complete medium was used to replace the transfection medium and the cells of each group were aliquoted in triplicate. After 24-h incubation, the transfected SGC7901 cells were harvested for protein extraction. Initially, the culture plates were placed on ice, followed by addition of 250 μL lysis buffer for 10 min. The lysed cells were scraped off and sonicated for 10 min. Subsequently, the cell lysates were centrifuged at 20000 g for 30 min, and the supernatant containing cell proteins was collected. The whole cell lysate proteins were purified using a Cleanup kit (ProteoPrep® Total Extraction Sample Kit, Sigma-Aldrich, United States) to remove salt and lipid impurities. Protein concentration was determined by bicinchoninic acid (BCA) method. Finally, 600 μg protein from each sample was used for 2D gel electrophoresis.
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