Total proteins were isolated from BT20 cells using RIPA reagent (Invitrogen). The proteins were subject to separating in a 10% SDS/PAGE gel and then transferred onto a poly(vinylidene difluoride) (PVDF) membrane (Millipore). The PVDF membrane was incubated with a blocking buffer (3% BSA) for 1 h at room temperature and then incubated with anti‐KLF4 [Cell Signaling Technology (CST), Danvers, MA, USA; rabbit mAb #12173; 1 : 1000] or anti‐GAPDH (CST; rabbit mAb #5174; 1 : 1000) primary antibodies overnight at 4 °C. Then, the PVDF membrane was incubated with the secondary antibodies (Zhongshan Biotechnology, Beijing, China; goat anti‐rabbit; 1 : 10 000) for 1 h at room temperature. Finally, the PVDF membrane was exposed by using a SuperSignal West Dura Extended Duration Substrate Kit (Thermo Fisher Scientific, Beijing, China).
Ripa reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
RIPA reagent is a detergent-based buffer used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of ionic and non-ionic detergents, as well as salts and buffers, to facilitate the efficient lysis of cells and the release of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Ecl system, by CWBIO (3 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Supersignal west dura extended duration substrate kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
36 protocols using ripa reagent
1
Western Blot Analysis of KLF4 and GAPDH
2
Western Blotting Procedure for Protein Analysis
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Western blotting analysis was carried out as detailed elsewhere (60 (link),62 (link)). Fish were sacrificed after anesthesia and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty micrograms of total proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)]. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was carried out as detailed previously (60 (link)).
3
Protein Quantification and Western Blot
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Total protein from each group was separated with RIPA reagent (Invitrogen, USA). Quantification was performed using a BCA kit. 10% SDS-PAGE was adopted to separate protein. The protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen, USA) by the semidry transmembrane method. Then, they were blocked using 5% skim milk for 2 h at 25. They were incubated overnight with appropriate amounts of the primary antibody (Absin, China) at 4°C. PVDF membranes were washed the next day using Tris-HCL with Tween composite buffer and then were incubated for 2 h with the secondary antibody (Cell Signaling Technology, USA). The expression of each protein was quantified by the gray scale values scanned with the Gel Imager.
4
Protein Expression Analysis in Chondrocytes
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The transfected chondrocytes were washed with cold PBS and lysed in RIPA reagent (Invitrogen, Carlsbad, CA, U.S.A.). The proteins were quantified using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, U.S.A.) and electrophoresed on a 10% SDS-PAGE gel (Beyotime Biotechnology). Then, the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, U.S.A.), and blocked with 5% nonfat milk for 2 h at 37°C. The membranes were incubated with primary antibodies against EIF4G2 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and GPAHD (Santa Cruz Biotechnology) at 4°C overnight. All primary antibodies were diluted in 1% bovine serum albumin (Beyotime Biotechnology) solution with a dilution of 1:2000. After the membranes were washed with TBST buffer, and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology). Signals of bands were recorded using ECL detection system (Pierce Biotechnology) according to manufacturer’s information. Data were analyzed using Quantity One software (Bio-Rad, Hercules, CA, U.S.A.).
5
Protein Quantification via Western Blot
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Total proteins were extracted from each sample using RIPA reagent (Invitrogen, USA) and then separated via polyacrylamide SDS gel and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, MA). The membranes were then incubated with the primary antibodies (Supplementary Table 1 ) and subsequently the secondary antibody. The proteins were quantified using an ECL system. Western blot analyses were performed in triplicates, and the target protein bands were quantified using scanning densitometry, Image-J software, and normalized to the signal intensity of GAPDH. The relative density (RD) of protein bands was analyzed via ImageJ software.
6
Mitochondrial Protein Expression Analysis
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Fish were sacrificed after anesthesia, and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty μg of total cellular proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. After transfer, the membrane was incubated in 1 × PBST (1 × PBS, 1% Tween 20) and 5% nonfat dry milk for 1 h. After blocking, the membrane was incubated with primary antibody overnight at 4°C following with secondary antibody. The hybridized membrane was then exposed to chemiluminescence reagent for 1 min and developed by ChemiScope 3300 mini (CLiNX, Shanghai). The antibodies were obtained from different companies including Mtu1 from Hangzhou HuaAn Biotecnology Co (HuaBio), Gapdh (SAB2701826) from Sigma-Aldrich, Nd1 (ab74257), Nd6 (ab81212), Atp5a (ab188107), Sdhb (ab151684) and Yars2 (ab127542) from Abcam, Cytb (A9762) from ABclonal, Co2 (55070-1-AP), Kars (14951-1-AP), Lars2 (17097-1-AP), Tufm (26730-1-AP), Tfb2m (24411-1-AP) and Tom20 (1802-1-AP) from Proteintech. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (30 (link),42 ).
7
Western Blot Analysis of Protein Expression
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Total protein in transfected PANC-1 and AsPC-1 cells at 48 h was lysed in ice-cold RIPA reagent (Invitrogen), and protein samples were split in aliquots. Equally, 25 μg proteins were subjected to the standard Western blotting procedures. The special primary antibodies were from Proteintech (Wuhan, China) including c-myc (cat. 10,828-1-AP, 1:5000), cyclin D1 (cat. 26,939-1-AP, 1:2000), E-cadherin (cat. 20,874-1-AP, 1:25,000), vimentin (cat. 10,366-1-AP, 1:10,000), and β-actin (cat. 20,536-1-AP, 1:5000). The gray density of protein bands was detected using Image J software (National Institutes of Health) to reflect relative protein expression with normalization to β-actin.
8
Western Blotting Protocol for Mitochondrial Proteins
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Western blotting analysis was performed as detailed elsewhere (42 (link),44 (link),49 (link)). Fish were sacrificed after anesthesia and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty micrograms of total cellular proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. The antibodies used include anti-Gtpbp3 (Sigma, HPA042158), anti-Nd5 (Abcam, ab92624), anti-Nd6 (Abcam, ab81212), anti-Cytb (ABclonal, A9762), anti-Co2 (Proteintech, 55070-1-AP), anti-Sdha (Proteintech, 14865-1-AP), anti-Atp5c (Proteintech 60284-1-Ig), anti-Tfam (Proteintech, 19998-1-AP), and anti-Tufm (Proteintech, 26730-1-AP), anti-Yars2 (Abcam, ab228957), anti-Kars (Proteintech, 14951-1-AP), anti-Afg3l2 (Proteintech, 14631-1-AP), anti-Clpp (Proteintech, 15698-1-AP) and anti-Gapdh (Sigma, SAB2701826). Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (42 (link)).
9
Protein Extraction and Western Blotting
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Total proteins were extracted from each sample using RIPA reagent (Invitrogen, United States) and then separated via polyacrylamide SDS gel and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, MA, United States). The membranes were incubated with the primary antibodies (Supplementary Table 1 ) and then the secondary antibody. Levels of proteins were determined using an ECL system.
10
Cardiac Gene and Protein Expression Analysis
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The RNAs of the heart cells were isolated and extracted with TRIzol (Invitrogen). Then, cDNAs were obtained by using Transcriptor First Strand cDNA Synthesis Kit (Roche). The amplification of gene sequences was then performed on LightCycler 480 SYBR Green Master Mix (Roche). The quantification results were analysed according to GAPDH gene expression. Shown in Table S1 of the supplementary materials are the primer details.
The proteins of cells and tissues were extracted by using RIPA reagent (Invitrogen), and the concentration of proteins was determined. For the separation of target proteins, 10% SDS‐PAGE was used. Then, the proteins in the gel were then transferred onto PVDF membranes (Millipore) and subsequently blocked with 5% non‐fat milk to remove possible interference from impurities. The corresponding antibodies were used to bind target proteins and incubated overnight at 4°C. After 1 hour incubation with the secondary antibodies at 37°C, the protein were reacted with ECL reagents (Bio‐Rad), and the proteins were scanned by using Bio‐Rad ChemiDoc XRS. The expression of the protein levels was standardized according to endogenous reference protein. Details on the primary antibodies used are provided in TableS2 in the supplementary materials.
The proteins of cells and tissues were extracted by using RIPA reagent (Invitrogen), and the concentration of proteins was determined. For the separation of target proteins, 10% SDS‐PAGE was used. Then, the proteins in the gel were then transferred onto PVDF membranes (Millipore) and subsequently blocked with 5% non‐fat milk to remove possible interference from impurities. The corresponding antibodies were used to bind target proteins and incubated overnight at 4°C. After 1 hour incubation with the secondary antibodies at 37°C, the protein were reacted with ECL reagents (Bio‐Rad), and the proteins were scanned by using Bio‐Rad ChemiDoc XRS. The expression of the protein levels was standardized according to endogenous reference protein. Details on the primary antibodies used are provided in Table
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