Radio immunoprecipitation assay was used for isolating proteins of ARPE-19 cells and a BCA Protein Quantification kit evaluated the concentration of proteins. Subsequently, protein samples (40 μg) were separated using 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China), followed by moving to polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). After membranes were sealed via skimmed milk for 1 h, they were incubated with following primary antibodies at 4 °C overnight: anti-Nrf2 (1:500, ab62352, Abcam), anti-HO-1 (1:2000, ab52947, Abcam), anti-GPX4 (1:2000, ab125066, Abcam) and anti-β-actin (1:200, ab115777, Abcam). Then the anti-rabbit secondary antibody (1:2000, ab205718, Abcam) was added to incubate for 1 h. Finally, the bands were examined using an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA).
Sodium dodecyl sulfonate polyacrylamide gel
Manufactured by Solarbio
Sourced in United States, China
Sodium dodecyl sulfonate-polyacrylamide gel is a laboratory equipment used for the separation and analysis of macromolecules, such as proteins, based on their molecular weight. It functions as a support matrix for electrophoresis, allowing the separation of molecules through the application of an electric field.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (10 mentions)
Polyvinylidene difluoride membrane, by Merck Group (8 mentions)
Ab181602, by Abcam (6 mentions)
Ab205719, by Abcam (5 mentions)
Bca protein assay kit, by Tiangen Biotech (5 mentions)
Bca protein quantification kit, by Vazyme (5 mentions)
Ab6789, by Abcam (4 mentions)
Bs 0295m hrp, by Bioss Antibodies (4 mentions)
Ab231303, by Abcam (4 mentions)
Ab16667, by Abcam (4 mentions)
Ecl kit, by Beyotime (3 mentions)
Polyvinylidene difluoride membrane, by Cytiva (3 mentions)
Bs 2188r, by Bioss Antibodies (3 mentions)
Ab49254, by Abcam (3 mentions)
Ab192615, by Abcam (3 mentions)
Ab113743, by Abcam (3 mentions)
Ab62194, by Abcam (3 mentions)
Ab133557, by Abcam (3 mentions)
Ecl western blot kit, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
17 protocols using sodium dodecyl sulfonate polyacrylamide gel
1
ARPE-19 Oxidative Stress Protein Analysis
2
Western Blot Analysis of Protein Markers
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Total RNA was obtained from tissues and cells by using RIPA buffer (Beyotime, Shanghai, China) and measured using a BCA protein assay kit (Tiangen, Beijing, China). Then 20 μg proteins were resolved by 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) and then transferred onto polyvinylidene di uoride membranes (Pall Corporation, New York, NYC, USA). Next, the samples were immersed with 5% skim milk for 1 h and incubated with primary antibodies: cyclin D1 (ab16663; Abcam), proliferating cell nuclear antigen (PCNA; ab92552; Abcam), TMEM106B (ab264323; Abcam) or GAPDH (ab181602; Abcam) at 4°C overnight followed by incubation with relevant secondary antibody (ab205719; Abcam) for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (Vazyme) was utilized to visualize the blots.
3
Western Blot Analysis of Extracellular Vesicle Proteins
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The extraction of total protein was done utilizing RIPA buffer (Beyotime) and quantified utilizing the BCA protein assay kit (Tiangen, Beijing, China). An equal amount of protein was resolved with sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich). After blocking in 5% defatted milk for 1 h at indoor temperature, the membranes were incubated with primary antibodies against CD9 (ab223052; Abcam, Cambridge, MA, USA), CD63 (ab68418; Abcam), CyclinD1 (ab226977; Abcam), Bax (ab104156; Abcam), matrix metalloprotein 13 (MMP13; ab39012; Abcam), aggrecan (ab36861; Abcam), TRAF6 (ab137452; Abcam) or GAPDH (ab37168; Abcam) overnight at 4 °C and indicated secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. The ECL kit (Beyotime) was employed for chemiluminescence imaging.
4
Protein Quantification and Western Blot
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The isolation of total protein was executed utilizing RIPA buffer (CWBio, Beijing, China) and the concentrations of proteins were measured with a BCA Protein Quantification Kit (Vazyme, Nanjing, China). Then equal amounts of protein were separated through sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis followed by transferring into polyvinylidene difluoride membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% non-fat milk for 1 h at indoor temperature, the membranes were incubated overnight with primary antibodies: β-actin (bs-0061R; Bioss, Beijing, China) and HMGB1 (bs-0664R; Bioss) at 4°C and probed with horseradish peroxidase-conjugated secondary antibody (bs-0295M-HRP; Bioss) for 1 h at indoor temperature. The signals were examined using chemiluminescent substrate kit (Millipore, Bedford, MA, USA).
5
Protein Quantification and Western Blot Analysis
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Total protein in GC cells was extracted utilizing RIPA buffer (Beyotime) and determined utilizing a BCA protein assay kit (Tiangen, Beijing, China). Then the equal amount of protein (30 μg) was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and blotted onto polyvinylidenedifluoride membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% defatted milk for 1 h, the membranes were cultivated overnight with primary antibodies against CyclinD1 (1:2,000; bs-0623R; Bioss, Beijing, China), E-cadherin (1:2,000; bs-10009R; Bioss), N-cadherin (1:2,000; bs-1172R; Bioss), or GAPDH (1:5,000; bs-2188R; Bioss) at 4°C followed by interaction with HRP-conjugated secondary antibody (1:5,000; bs-0295M-HRP; Bioss) for 1.5 h at room temperature. The protein bands were visualized with an ECL reagent (Vazyme, Nanjing, China) and analyzed via ImageJ v1.8.0 (National Institutes of Health).
6
Western Blot Analysis of Protein Expression
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Total protein was extracted with radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO, USA) and quantified with bicinchoninic acid protein assay kit (Tiangen, Beijing, China). Following separation by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) electrophoresis, the proteins were blotted to polyvinylidene difluoride membranes (Sigma-Aldrich). After that, the membranes were blocked for 1 h with 5% non-fat milk and cultivated overnight with primary antibodies against APPBP2 (bs-11639R; Bioss, Beijing, China), Vimentin (bs-0756R; Bioss), E-cadherin (bs-1519R; Bioss), N-cadherin (bs-1172R; Bioss), and GAPDH (bs-2188R; Bioss) at 4°C. Thereafter, the membranes were kept with horseradish peroxidase-conjugated secondary antibody (bs-0294M-HRP; Bioss) for 2 h at indoor temperature. The immunoblots were visualized by using enhanced chemiluminescence reagent (Beyotime, Shanghai, China).
7
Protein Isolation and Western Blot Analysis
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Total protein was isolated from serums, cells and exosomes using RIPA buffer (Beyotime) and quantified using BCA protein assay kit (Tiangen, Beijing, China). Then, equal amount of proteins was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, New York, NYC, USA). After being blocked with skim milk for 2 h, membranes were incubated with primary antibodies against multidrug resistance-associated protein 1 (MRP1; ab32574; Abcam, Cambridge, MA, USA), multidrug resistance protein 1 (MDR1; ab170904; Abcam), REV3L (ab111729; Abcam) or GAPDH (ab181602; Abcam) overnight followed by incubation with corresponding secondary antibody (ab150077; Abcam) for 2 h. The proteins were visualized using the enhanced chemiluminescence detection kit (Vazyme, Nanjing, China).
8
Quantitative Western Blot Analysis of Multidrug Resistance Proteins
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The tissues and cells were lysed in RIPA buffer (Beyotime) to extract the total protein. The extracted proteins were examined with a BCA Protein Quantification Kit (Beyotime). Next, 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) was employed to separate the proteins, which were then blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After 1 h of blockage in 5% skim milk, the membranes were probed with primary antibodies overnight at 4°C and then cultivated with the relevant secondary antibody (ab6728; Abcam, Cambridge, MA, USA) at indoor temperature for 2 h. The bands were visualized utilizing an ECL kit (Beyotime). All primary antibodies including multidrug resistance protein 1 (MRP1; ab32574), P-glycoprotein (P-gp; ab170904), lung-resistance-related protein (LPR; ab97311), EZH2 (ab186006) and β-actin (ab8224) were purchased from Abcam.
9
Protein Extraction and Western Blot Analysis
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Protein extraction was finished using RIPA buffer (CWBio, Beijing, China). The protein concentrations were examined via a BCA Protein Quantification Kit (Vazyme). Then, an equal amount of proteins was separated through sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk for 1 h at indoor temperature and immunoblotted with primary antibodies against hexokinase2 (HK2; bs-9455R; Bioss, Beijing, China), CPEB4 (bs-14020R; Bioss) and β-actin (bs-0061R; Bioss) overnight at 4°C followed by interaction with indicated horseradish peroxidase-conjugated secondary antibody (bs-0295M-HRP; Bioss) for 1 h. The immunoreactive bands were developed using ECL kit (Beyotime, Shanghai, China) and analyzed using Image J software.
10
Protein Extraction and Western Blot Analysis
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Protein extraction was finished using RIPA buffer (Beyotime) and protein concentration determination was executed utilizing a BCA Protein Quantification Kit (Vazyme). 20 μg proteins were separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) electrophoresis. Next, the proteins were blotted onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Afterward, the membranes were blocked utilizing 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against GAPDH (bs-2188R; 1:5,000; Bioss, Beijing, China), B-cell lymphoma-2 (Bcl-2; 1:2,000; bs-34012R; Bioss), BCL2-Associated X (Bax; 1:2,000; bs-0127R; Bioss), cleaved caspase-3 (bs-0081R; 1:2,000; Bioss), and PDE7B (bs-11576R; 1:2,000; Bioss) overnight at 4°C and secondary antibody (bs-0295M-HRP; 1:5,000; Bioss) for 1 h at room temperature, followed by ECL detection.
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