Cells were lysed with a RIPA buffer (CW2333S, CWbio, China) and a protease inhibitor cocktail set I (539131, Millipore, USA). The cellular proteins' supernatants were collected by centrifugation at 14,000 g for 20 min at 4°C. Protein concentrations were measured with the BCA protein assay kit (CW0014S, CWbio). Equal amounts of proteins were separated by 10% SDS-PAGE (P0014B, CWbio) and were transferred to PVDF membranes (ISEQ00010, Millipore, USA), which were blocked for 1 h with 5% bovine serum albumin (0332, Amresco) and were incubated overnight at 4°C with related primary antibodies, including LDHA (1:1,000, C4B5, Cell Signaling) matrix metalloprotein 2 (MMP2, 1:1,000, ab37150, Abcam), ZO1 (TJP1 tight junction protein 1; 1:1,000, D7D12, Cell Signaling), E-cadherin (1:1,000, 24E10, Cell Signaling), N-cadherin (1:1,000, D4R1H, Cell Signaling), Vimentin (1:1,000, D21H3, Cell Signaling), Slug (1:1,000, C19G7, Cell Signaling), and β-ACTIN (1:1,000, D6A8, Cell Signaling). After probing with secondary antibodies (GK600510">GK600510 , Gene Tech, China), the membranes were conjugated to horseradish peroxidase (HRP) in room temperature for 1 h, and the bands were exposed by a chemiluminescent HRP substrate (WBKLS0500, Millipore, USA). The quantitative analysis of Western blots was carried out by Image J (https://imagej.nih.gov/ij/download.html ).
Cw2333
Manufactured by CWBIO
Sourced in China
The CW2333 is a high-precision spectrophotometer designed for accurate measurements of optical density and absorbance. It features a wide wavelength range and advanced optics to provide reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Cw2200, by CWBIO (8 mentions)
Iseq00010, by Merck Group (6 mentions)
Cw0014, by CWBIO (5 mentions)
Ipvh00010, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Wbkls0500, by Merck Group (2 mentions)
Ab126605, by Abcam (2 mentions)
Ab195377, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Cw0103, by CWBIO (2 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Chemiscope 6000, by Clinx (2 mentions)
A18817, by ZSGB-BIO (2 mentions)
Mab73601 100, by R&D Systems (2 mentions)
Cw0022, by CWBIO (2 mentions)
P0010, by Beyotime (2 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab37150, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Gk600510, by Gene Tech (1 mentions)
33 protocols using cw2333
1
Protein Expression Analysis by Western Blot
2
Protein Extraction Using RIPA Lysis Buffer
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Radioimmunoprecipitation assay Lysis Buffer (strong; CWBIO CW2333S) was mixed with protease inhibitor cocktail (100×; CWBIO CW2200) in a ratio of 1:99 for the preparation of 1× extraction reagent for protein extraction. After the treated cells were rinsed with PBS, 1× protein extraction reagent was placed on ice. The lysates were transferred to new centrifuge tubes and incubated on ice for 20 minutes. They were then centrifuged at 14 000×g for 10 minutes before the supernatants were transferred to new tubes. The final extracted samples were kept at −80 °C.
3
Western Blot Analysis of Kidney Proteins
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Kidney tissue and HK-2 cells were harvested and lysed with RIPA lysis buffer (CW2333S, CWbio) containing a protease inhibitor cocktail (CW2200, CWbio). A BCA protein assay kit (BL521A, Biosharp) was used to determine the concentration. An equivalent quantity of protein was separated on a 7.5 ~ 12% SDS-PAGE gel (1610173, Bio-Rad) and transferred to PVDF membranes (ISEQ00010, Millipore). The membranes were blocked and probed with various primary antibodies overnight at 4 °C. After incubation, HRP-conjugated secondary antibody was used at room temperature for 1 h. Primary antibodies are shown in supplementary table 3 . Proteins were visualized using enhanced chemiluminescence (WBULS0100, Millipore) and quantified using ImageJ software.
4
Hippocampal SynCAM1 and PV Expression
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Hippocampus tissues were harvested at ages P14, 21, 28, 35, and 42 and then homogenized using RIPA lysis buffer (CW2333s, CW, biotech), supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF)‐protease inhibitors (CW2200S, CW, biotech). Protein concentration was determined by a BCA protein assay kit (GK10009, GLPBIO). Equal amounts of protein samples (20 μg) were loaded and separated by 10% or 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (G2037‐50T, Servicebio) transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 3% non‐fat milk for 1 h at room temperature, membranes were incubated at 4°C overnight with primary antibodies of chicken anti‐SynCAM1(1:1000, CM004‐3, MBL), rabbit anti‐PV (1:500, A13538, ABclonal), or rat anti‐β‐actin (1:10000, 660009‐1, Proteintech). After washing with Tris Buffer Saline with Tween‐20 (TBST), the membranes were incubated for 2 h at room temperature with secondary antibodies of goat anti‐rabbit IgG (1:10,000, SA00001‐2, Proteintech), goat anti‐mouse IgG (1:10,000, SA00001‐2, Proteintech), and goat anti‐chicken IgG (1:5000, L35001, Signalway) and visualized with an enhanced chemiluminescent detection (ECL) kit (WBKLS0100, Millipore) and analyzed with Image J software.
5
Western Blot Analysis of m6A Regulators
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Cells were fully lysed with ice-cold strong RIPA lysate (CW2333S, CWBIO) on ice for 10 min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C to remove impurities. The supernatants after centrifugation were the protein solutions, and protein concentrations were quantified by BCA protein assay kit (CW0014S, CWBIO). Equal amounts of proteins from each group were electroblotted and separated by SDS-PAGE and electrotransferred onto PVDF membranes (0.45 μm, IPVH00010; 0.2 μm, ISEQ00010; Millipore). After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with primary antibodies including DIRAS1 (ab65139, Abcam), METTL3 (ab195352), METTL14 (ab220030), ALKBH5 (ab195377), FTO (ab126605) and GAPDH (ab8245). Subsequently, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (RPN2105, Amersham), and the gray values of the bands were read by Image J software.
6
Western Blot Protocol for Protein Analysis
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Prepared cells were dissolved in 100 µL RIPA lysis buffer (CW2333S, CWBIO) including 100× protease inhibitor (CW2200, CWBIO). After centrifugation, the lysates were denatured for 5 min in 5× SDS-PAGE loading buffer (P0015, Beyotime). Proteins were separated on an SDS-PAGE gel and transfered to a PVDF membrane. Next, we block it with 5% skimmed milk powder at 4 °C overnight. After that, the membrane was incubated with primary antibodies for 1 h, then the corresponding secondary antibodies at room temperature for 0.5 h. Developed membranes were imaged using an Azure c300 digital imager system (Azure Biosystems, Dublin, CA).
7
Western Blot Analysis of Protein Expression
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Tissue samples or cultured cells were lysed in RIPA buffer (CWBIO, CW2333S), and the protein concentration was measured by the BCA protein assay. Equal amounts of protein were resolved by 10% or 15% SDS–PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked at room temperature with 5% skimmed milk and then incubated with primary antibodies overnight at 4 °C. Primary antibodies against YES1 (1:1000, ABclonal, A0628), LC3b (1:1000, Abcam, AB192890), and Gapdh (1:5000, Proteintech, 51067-2-AP) were used. After washing with TBST (Tris-buffered saline with Tween), the membranes were further incubated with fluorescent secondary antibodies (1:5000, ABclonal, AS014) at room temperature in the dark for 1-2 h. Then, the membranes were detected by an enhanced chemiluminescence detection kit (Epizyme, SQ202). GAPDH was used as internal control. The signals were detected using an Odyssey detection system (Odyssey CLx, LI-COR biosciences, NE, USA). Quantification analysis of western blots was performed using ImageJ software (Bethesda, USA).
8
Quantitative Proteome Analysis of LMC-Treated Tissues
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Total protein from LMC-treated tissues was extracted using lysis buffer (CW2333S, Cwbiotech, Beijing, China) following the manufacturer's protocol. The total proteins from NGM, AH, GPDAC and LMGAC tissue samples were labeled with different iTRAQ reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Four labeled lysates were mixed and lyophilized before they were desalted. Then, the samples were dissolved in deionized water containing 0.1% formic acid (FA, Tedia Company, Fairfield, OH, USA) and eluted three times with Sep-Pak C18 1 cc Vac cartridges (Waters Corporation, Milford, MA, USA). The cleaning solution was collected and lyophilized to obtain the final samples.
9
Quantitative Protein Analysis Protocol
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The total protein was extracted by RIPA lysis buffer (CWBIO, CW2333S) mixed with cocktail inhibitors on ice, and protein concentration was quantified with BCA assay kit (CWBIO, CW0014S) according to manufacturer's instructions. Protein was separated by SDS‐PAGE gel and then transferred to PVDF membranes (Millipore, ISEQ00010), and then the membranes were incubated with 5% skimmed milk in TBST and hybridized with primary antibodies overnight at 4 °C. After incubated with secondary antibodies at room temperature, and protein was detected using chemiluminescence detection reagent (Millipore, WBKLS0500), and the bands were analyzed with imaging system (Syngene). The primary antibodies were anti‐FOSL1 (Abcam, ab232745) and anti‐Tubulin (Abcam, ab7291).
10
Western Blot Analysis of Cellular Proteins
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Cells were lysed on ice with ice-cold strong RIPA buffer (CW2333S, CWBIO) for 10 min. The lysate was centrifuged at 12,000g for 10 min at 4 °C to remove precipitates, such as cell debris, and the protein was obtained in the supernatant. Protein concentration was determined by BCA assay kit (CW0014S, CWBIO). Protein samples were boiled for denaturation, and 1 × loading buffer was added. Subsequently, 20 μg of protein in each sample was subjected to SDS-PAGE loading and electrophoresis for separation (spacer gel, constant voltage 80V; 12 % separating gel, constant current 30 mA) based on molecular weight size. Then, the proteins were electrotransferred onto (constant voltage 110V, 90 min) a PVDF membrane (0.45 μm, IPVH00010; Millipore). The membrane was incubated with 5 % skim milk for 1 h at room temperature for non-specific antigen blocking, and incubated with primary antibody solution (anti-PSMA7, 1:1000, 67817-1-Ig, Proteintech; anti-GAPDH, 1:5000, ab8245, Abcam) overnight at 4 °C and with the corresponding secondary antibody for 2 h at room temperature. The bands were subjected to chemiluminescence using Enhanced Chemiluminescence reagent (RPN2105, Amersham).
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