Using RIPA buffer (R0020, Beijing Solarbio Science & Technology Co.,Ltd., China) and Phenylmethylsulfonyl fluoride (PMSF, P0100, Beijing Solarbio Science & Technology Co.,Ltd., China), proteins were extracted from all cell lines (NCM460, Caco-2, SW480, DLD-1, HCT 116), colon tissue of human and animal. BCA Protein Assay kit (PA115-01, TIANGEN BIOTECH BEIJING CO., Ltd., China) for protein quantification. Protein samples were transferred to nitrocellulose (NC) membranes (GE Healthcare Life Science, Pittsburgh, USA) by SDS-PAGE gel (G2043, Wuhan Servicebio Technology Co., Ltd., HuBei) at 10% concentration. The membranes were blocked with 5% skim milk powder for 1.5 h, then incubated with primary antibody CHKB (1:1000, PH5354, Abmart) and PEMT (1:1000, PK41366, Abmart) overnight at 4°C. The membrane was incubated with HRP Conjugated AffiniPure Goat Anti-rabbit IgG (BA1055, Boster Biological Technology Co., Ltd., Wuhan) for 1 h at room temperature. The membranes were detected using SuperSignal West Pico PLUS (34580, Thermo Fisher Scientific) and visualized by iBright CL1500 Imaging System (Thermo Fisher Scientific).
R0020
Manufactured by Solarbio
Sourced in China
R0020 is a laboratory centrifuge designed for general-purpose use in research and diagnostic applications. It is capable of separating and concentrating various biological samples, such as cells, organelles, and macromolecules, based on their sedimentation rate. The centrifuge is equipped with a rotor that can accommodate multiple sample tubes, allowing for efficient processing of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (3 mentions)
P1040, by Solarbio (2 mentions)
β actin, by Wuhan Servicebio Technology (2 mentions)
Sa00001 1 2, by Proteintech (2 mentions)
P1260, by Solarbio (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose nc membrane, by GE Healthcare (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Boster Bio (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Bicinchoninic acid protein assay kit, by Solarbio (1 mentions)
Cw2200, by CWBIO (1 mentions)
Cw2383, by CWBIO (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Pentobarbital sodium, by Solarbio (1 mentions)
Xylazine hydrochloride, by Merck Group (1 mentions)
Fibronectin, by Abcam (1 mentions)
22 protocols using r0020
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Osteogenic Markers
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The cells were lysed using RIPA buffer (R0020; Solarbio) with phosphatase inhibitors (CW2383; Cwbio) and protease inhibitor (CW2200; Cwbio). Proteins extracted from each sample were determined using bicinchoninic acid protein assay kit (PC0020; Solarbio). Heat-denatured proteins were separated by 10% SDS-PAGE and transferred onto a 0.22 μm polyvinylidene difluoride membrane (IPVH00010; Millipore). The membranes were blocked in 5% skim milk at room temperature for 1 h; then, each membrane was incubated with antibodies against Traf6, Nfatc1, Ctsk, Runx2, ALP, Col1α1, and HRP-β-actin at 4°C overnight. After the membranes were incubated with the respective secondary antibodies for 1 h at room temperature, the blots were detected using a chemiluminescent HRP substrate (wbkls0500; Millipore). We used the ImageJ software to quantify the grayscale value of bands.
3
HSV-1 Infection Model in CD-1 Mice
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Six-week male CD-1 (ICR) mice were purchased from Shanghai Laboratory Animals Center. For HSV-1 infection, mice were anesthetized by intraperitoneal injection of 0.4 ml of a mixture consist of 500 ug/ml xylazine hydrochloride (Sigma, X1251) plus 4 mg/ml pentobarbital sodium (Solarbio) in sterile saline. Then 2 x 105 pfu of virus was added onto each scarified cornea in 3 μl of PBS. For TG acquisition, mice were euthanized by cervical dislocation while under anesthesia with isoflurane and TG were collected into the lysis buffer and placed on dry ice before RNA extraction and placed into -80°C as described previously [59 (link)]. For western blot analysis, we homogenized TG in lysis buffer (Solarbio, R0020) and extracted protein according to the manufacturer’s protocol. For determination of viral titers in TG, TG were homogenized in cell culture media for titer determination. For eye swab collection, mice were anesthetized in an induction chamber with isoflurane (3% in oxygen 0.5 ml/min) using a V1 Table Top anesthesia machine (Colonial Medical Supply). Both eyes of each mouse were swabbed with cotton-tipped applicators, suspended in 1 ml of cell culture medium.
4
Western Blot Analysis of Cell Lysates
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Cell or tissue lysis was carried out with RIPA buffer (R0020; Solarbio, China), and total protein amounts were determined with the BCA kit (PC0020; Solarbio). After addition of the corresponding loading buffer (P1040 or P1019; Solarbio), the mixture underwent a 10-min boiling step. Equal amounts of total protein were resolved by SDS-PAGE and electro-transferred onto PVDF compound membranes (Millipore, USA) treated with methanol. Upon blocking with 5% non-fat milk, the membranes underwent overnight incubation (4° C) with primary antibodies raised against CDX2 (60243; Proteintech, 1:1000), CFTR (2784; Abcam, 1:1000), active β-catenin (8814; CST, 1:1000), Snail (3879; CST, 1:1000), E-cadherin (14472; CST, 1:1000), Vimentin (5741;CST, 1:1000), Collagen type III (Col-III; 22734; Proteintech, 1:1000, Fibronectin (2413; Abcam, 1:1000) and β-actin (Pumei, China, 1:4000), respectively. Then, secondary antibodies were added at ambient for 1h. Finally, the ECL solution was added, and a Bio-Rad gel imaging system (Bio-Rad, USA) was employed for analysis.
5
Co-Immunoprecipitation Workflow for RASD2 Protein
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Co-IP was performed by the recommended protocol of kit manufacturer (Abs955, Absin,
Shanghai, China). Firstly, RIPA buffer (R0020, Solarbio) with 1% PMSF solution was added
to the collected tissue, and the tissue was homogenized by homogenizer. Then, the samples
were centrifuged at 12 000 rpm for 20 minutes at 4°C, and the supernatant was removed for
use. Primary antibody (RASD2, RHES-101AP, Fabgennix, Frisco, TX, USA) was added to the
samples, while homologous antibodies (Rabbit IgG, abs20035, Absin) from nonspecific
immunization were used as controls and the samples were incubated overnight at 4°C.
Protein A and G were added to the samples and gently mixed overnight at 4°C then
centrifuged at 12 000 rpm for 1 minute to retain the precipitate. Precipitate was washed
by wash buffer 3 times. 1*SDS sample buffer was added to resuspend the precipitate, and
the sample was held at 95°C–100°C for 5 minutes. All samples were subsequently analyzed by
WB.
Shanghai, China). Firstly, RIPA buffer (R0020, Solarbio) with 1% PMSF solution was added
to the collected tissue, and the tissue was homogenized by homogenizer. Then, the samples
were centrifuged at 12 000 rpm for 20 minutes at 4°C, and the supernatant was removed for
use. Primary antibody (RASD2, RHES-101AP, Fabgennix, Frisco, TX, USA) was added to the
samples, while homologous antibodies (Rabbit IgG, abs20035, Absin) from nonspecific
immunization were used as controls and the samples were incubated overnight at 4°C.
Protein A and G were added to the samples and gently mixed overnight at 4°C then
centrifuged at 12 000 rpm for 1 minute to retain the precipitate. Precipitate was washed
by wash buffer 3 times. 1*SDS sample buffer was added to resuspend the precipitate, and
the sample was held at 95°C–100°C for 5 minutes. All samples were subsequently analyzed by
WB.
6
SDS-PAGE and Western Blot Analysis
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Proteins were separated by adding protease inhibitor (Kangway, CW2200S) in ice-cold RIPA buffer (Solarbio, R0020) and protein concentration was determined by bicinchoninic acid assay (BCA, Beyotime, P0012). PAGE Gel Rapid Preparation Kit (15%) Polyacrylamide Gelatins were prepared (EpiZyme, PG114), proteins were electrophoresed, transferred to a PVDF membrane (polyvinylidene difluoride membrane) and detected with primary and secondary antibodies. Primary antibodies used: RPS17 (Sinobiological, 202778-T46), β-actin (Servicebio, A2317). Protein bands detected by the antibodies were visualized by enhanced chemiluminescence (Beyotime, P0018FM-2) and evaluated using Image J. The antibodies were used to detect the protein bands.
7
Quantitative Western Blot Analysis of ARID5B Protein
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Cells were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), and protein levels were quantified using a BCA protein assay kit (P0012S, Beyotime). The extracted cellular proteins were mixed with loading buffer (v/v = 4:1) (P1040, Solarbio) and boiled for 10 min. Protein samples (30 μg per well) were separated by 8% SDS–PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with 5% BSA in 1 × TBST (0.2% Tween-20, pH = 7.6) buffer for 2 h and then incubated with primary antibodies against GAPDH (1:100,000, A19056, ABclonal, Wuhan, China) and ARID5B (1:2000, NBP1-83622, Novus, Shanghai, China) at 4 °C overnight. After being washed with 1 × TBST buffer four times (10 min each time), the membranes were then incubated with HRP-conjugated mouse anti-rabbit (1:5000, AS061, ABclonal) and goat anti-mouse (1:5000, AS003, ABclonal) secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using an ECL reagent (PE0010, Solarbio) and photographed by a Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Image-Pro Plus (v 6.0) software was used to perform the densitometric semiquantitative analysis of protein bands. The protein expression was normalized against GAPDH.
8
Quantitative Protein Expression Analysis
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Cells were added with RIPA lysate (R0020; Solarbio) and protease inhibitor PMSF (P0100; Solarbio) with a final concentration of 1 mM, lysed and centrifuged at 15,000 x g for 10 min at 4˚C to extract the cell protein. The protein concentration was quantified by BCA method, and the final protein concentration was adjusted. The sample protein concentration was 5-10 µg/µl with 10 µl applied to each well, and the gel was electrophoresed using an 8% SDS-PAGE separation gel. After electrophoresis, the protein was transferred to the PVDF membrane by a semi-wet transfer method. 5% Skim milk powder was blocked for 1 h, and then Toll-like receptor (TLR)4 (1:2,000, 66350-1-Ig; Proteintech), TLR7 (1:500, 17232-1-AP; Proteintech), GAPDH (1:5,000, 60004-1-Ig; Proteintech) were added. The primary antibody dilution solution was left at 4˚C overnight, then it was washed three times with 0.1% PBST. The corresponding HRP-labeled goat anti-rabbit (mouse) immune secondary antibody (1:3,000, SA00001-1/2; Proteintech) was added, then incubated at room temperature for 1 h. Then washed 3 times with 0.1% PBST. ECL chemiluminescence solution (PE0010; Solarbio) was added, after that, it was developed, fixed and images were taken in a dark room (LAS 4000; ImageQuant) for recording. ImageJ software was used for image analysis.
9
Western Blot Analysis of Kidney Proteins
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Total proteins of frozen kidney tissues and harvested cells were extracted with radioimmunoprecipitation buffer (R0020, Solarbio) with a protease inhibitor cocktail (P6730, Solarbio). After quantification with a BCA protein assay kit (No. 23227, Thermo Fisher), protein (20 μg) was separated by 8% or 10% SDS-PAGE and then transferred to a PVDF membrane. The membranes were blocked in 5% non-fat milk at room temperature (RT) for 1 h and then incubated with primary antibodies overnight at 4°C. Primary antibodies used were shown below: anti-Klotho (ab181373, Abcam; sc-515942, Santa Cruz), anti-DNA methyltransferase (DNMT) 1 (#5032, CST), anti-DNMT3a (#3598, CST), anti-DNMT3b (#67259, CST), anti-p-STAT3 (ab76315, Abcam), anti-α-smooth muscle actin (α-SMA; #14968, CST), anti-E-cadherin (E-cad; #14472, CST), and anti-GAPDH (ab181603, Abcam). The next day, membranes were incubated with HRP-labeled secondary antibodies (sc-2357 & sc-2005, Santa Cruz) for 1 h at RT. Immunoblot signals were detected and then analyzed by Image J software with normalization to GAPDH.
10
Protein Extraction and Immunoblotting Protocol
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Cells and tissues were lysed using RIPA buffer (R0020; Solarbio, Beijing, China), to which a mixture of protease phosphatase inhibitors (P1261; Solarbio, Beijing, China) was added. We used β-actin (1:1000, GB12001; Servicebio, Wuhan, China) primary antibodies as an internal reference. All antibody information used in this study is listed in Additional file 1 : Table B1. The electrophoretic protein was transferred to a PVDF membrane. We then blocked the membranes in 5% skim milk for 1 h and incubated them overnight at 4 °C with primary antibodies. Next, the membranes were washed three times and incubated with goat anti-rabbit secondary antibodies (BA1054; BOSTER, Wuhan, China) conjugated with horseradish peroxidase (HRP) for 2 h. Finally, the protein bands were detected by P2300 (NCM Biotech, Suzhou, China). Images were captured using ImageQuant LAS 500 (GE, Boston, America).
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