Cells were resuspended, centrifuged, and the cell pellet was collected. Add Protease Inhibitor Cocktail(ROCHE) and cell protein extraction reagent, repeatedly beat upon with a pipette to ensure complete cell lysis. After centrifugation at 13,000 g for 5 min at 4 °C, the supernatant was collected, which was the total protein solution. The sample protein concentration was determined using a BCA protein concentration assay kit (Aspen, Guangzhou, China), and ensure that the total amount of protein in each sample is 40 ug. Separation of proteins by SDS-PAGE (Aspen, Guangzhou, China) electrophoresis. And transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Add primary and secondary antibodies for antibody incubation. The freshly prepared ECL (Aspen, Guangzhou, China) mixed solution was added dropwise to the protein side of the membrane, and exposed in a dark room. The film was scanned and archived, and the optical density of the target bands was analyzed by the AlphaEaseFC software processing system.
Sds page
Manufactured by Aspen
Sourced in China
SDS-PAGE is a laboratory technique used for the separation and analysis of proteins based on their molecular weight. It involves the use of sodium dodecyl sulfate (SDS) to denature proteins and an electric field to separate them within a polyacrylamide gel.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Radioimmunoprecipitation assay buffer, by Aspen (2 mentions)
Bca protein concentration assay kit, by Aspen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Bca assay kit, by Roche (1 mentions)
Hrp 66009, by Proteintech (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Aspen (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Aspen (1 mentions)
Bicinchoninic acid (bca), by Aspen (1 mentions)
Rabbit anti phosphorylated erk, by Cell Signaling Technology (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase, by Abcam (1 mentions)
Alphaeasefc, by Bio-Techne (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
7 protocols using sds page
1
Protein Extraction and Western Blot Analysis
2
Renal iNOS and NF-κB Protein Quantification
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Renal iNOS and NF-κB p65 protein level were measured using Western blot analysis. In brief, renal tissue was completely homogenized in buffer. The supernatant was collected to extract total protein. Equal amounts of homogenate protein were separated by SDS-PAGE (ASPEN, Wuhan, China) and then transferred onto a nitrocellulose membrane. The membranes were blocked and incubated with primary antibodies (anti-NOS, Abcam; anti-NF-κB p65, Abcam; and anti-β-actin, Abcam) at 4°C overnight. The membranes were washed fully in TBST and incubated with anti-rabbit secondary antibody (Aspen). Finally, the relative protein expression levels were standardized to the level of β-actin and quantified using image analyzer software (AlphaEase FC, USA).
3
Mitochondrial Dynamics in Retinal Tissues
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Retinal tissues were lysed in radioimmunoprecipitation assay buffer (ASPEN, China) containing a protease inhibitor cocktail (ROCHE). All samples were subjected to protein content determination by using a BCA assay kit (ROCHE), electrophoresed by using SDS-PAGE (ASPEN, China), and transferred to a PVDF membrane. Subsequently, the membrane was blocked with 5% skimmed milk at room temperature for 1 h, incubated with the primary antibody diluent at 4 °C overnight, then incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blot was developed by using enhanced chemiluminescence and photographed in a dark room. Immune response bands were analyzed by utilizing Image J, and GAPDH and β-actin were used as the loading controls. The primary antibodies were as follows: Bax (1:1000, Abcam: ab182773), Bcl-2 (1:1000, Proteintech: 60178-1-Ig), cleaved caspase-3 (1:1000, CST: 9661), Bcl-xL (1:1000, CST: 2764); OPA1 (1:1000, CST: 80,471); DRP1 (1:1000, CST: 8570); MFN2 (1:1000, CST: 9482); GAPDH (1:1000, CST: 5174); and β-actin (1:1000, Proteintech: HRP-66,009).
4
Western Blot Analysis of Autophagy Proteins
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Briefly, retinal tissues and cultured cells were lysed in radio-immunoprecipitation assay buffer (ASPEN, China) containing protease inhibitor cocktail (ROCHE). All sample extracts were electrophoresed by SDS-PAGE (ASPEN, China) and electrophoretically transferred to PVDF membranes. The membranes were then blocked with 5% skim milk at room temperature for 1 h, incubated with primary antibody diluent at 4°C overnight and incubated with horseradish peroxidase-coupled secondary antibodies. The blots were developed using enhanced chemiluminescence (ECL) and the signals were captured in a dark room. The immunoreactive bands were analyzed in triplicate by ImageJ, with GAPDH being used as a loading control.
Primary antibodies were as follows: anti-LC3 (1:1,000, Cell Signaling Technology/CST: 3868), anti-Bax (1:1,000, CST: 2774), anti-Bcl-2 (1:1,000, CST: 9942), anti-casepase-3 (1:1,000, CST: 9662), anti-p62 (1:1,000, CST: 8025), anti-Beclin-1 (1:2000, CST: 3738), anti-PINK1 (1:2000, CST: 6946), anti-Parkin (1:2000, CST: 4211), anti-Optineurin (1:2000, CST: 58981), anti-DNP52 (1:2000, CST: 60732), anti-BNIP3L/Nix (1:2000, CST: 12396), anti-BNIP (1:2000, CST: 44060), and anti-GAPDH (1:1,000, CST: 8884).
Primary antibodies were as follows: anti-LC3 (1:1,000, Cell Signaling Technology/CST: 3868), anti-Bax (1:1,000, CST: 2774), anti-Bcl-2 (1:1,000, CST: 9942), anti-casepase-3 (1:1,000, CST: 9662), anti-p62 (1:1,000, CST: 8025), anti-Beclin-1 (1:2000, CST: 3738), anti-PINK1 (1:2000, CST: 6946), anti-Parkin (1:2000, CST: 4211), anti-Optineurin (1:2000, CST: 58981), anti-DNP52 (1:2000, CST: 60732), anti-BNIP3L/Nix (1:2000, CST: 12396), anti-BNIP (1:2000, CST: 44060), and anti-GAPDH (1:1,000, CST: 8884).
5
Protein Extraction and Western Blot Analysis
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Samples from each group of cells were collected and the total protein of cells was extracted using RIPA buffer (Aspen, USA) containing phosphatase inhibitor, then the concentration of total protein was determined using a BCA kit. Equivalent proteins were transferred to PVDF membranes after resolving them using SDS-PAGE (Aspen), then the membranes were incubated with primary antibodies overnight at 4°C. The membrane was then incubated with a secondary antibody (1:5,000) for 30 minutes at room temperature, and specific bands were detected using enhanced chemiluminescence reagents (ECL; Aspen, USA). The β-actin was used as an endogenous reference. Relative protein expression was calculated using ImageJ analyses (National Institutes of Health, Bethesda, MD, USA) of the band grayscales.27 (link),29
6
Tumulosic and Poricoic Acid Effects on SKOV3 Cells
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SKOV3 cells were seeded onto 6-well plates and treated with tumulosic acid or poricoic acid A respectively (0, 30, 50, and 80 μg/ml). Total protein was obtained and centrifuged at 13,000 g and 4 °C for 5 min. A bicinchoninic acid assay kit (ASPEN Biotechnology Co. Ltd, Wuhan, China; AS1086) was employed to detect the concentration of total protein. Proteins were separated using SDS-PAGE (ASPEN Biotechnology Co. Ltd, Wuhan, China; AS1012) and the separated proteins were transferred to a PVDF membrane at 300 mA. The membrane was blocked with 5% skimmed milk in PBS plus 0.1% Tween 20 (PBST) for 60 min, incubated with primary antibodies overnight at 4 °C: PI3K (CST, #4255), p-AKT (CST, #4060), AKT (CST, #9272), BCL2L1 (CST, #2764), BCL-2 (Abcam, ab196495) and GAPDH (Abcam, ab181602). Then incubated with goat anti-rabbit horseradish peroxidases (ASPEN Biotechnology Co. Ltd, Wuhan, China; 1:10,000, AS1107) for 30 min at room temperature. Finally, protein band development was performed. The film was scanned and archived, and the AlphaEaseFC software processing system analyzes the optical density values of the target band.
7
Protein Analysis of Wound Tissue
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Total protein was isolated from the wound tissue obtained at indicated times with RIPA (Aspen Inc.) following the manufacturer’s instruction. The protein concentrations were determined using the BCA (Aspen Inc.). Equal amounts of protein from each tissue were separated by SDS-PAGE (Aspen Inc.), transferred to a PVDF membrane, and incubated with 5% milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with rabbit anti-phosphorylated-ERK (1:1,000; Cell Signaling Technology), rabbit anti-ERK (1:2,000; Cell Signaling Technology), rabbit anti-phosphorylated-AKT (1:1,000; Cell Signaling Technology), rabbit anti-AKT (2:1,000; Cell Signaling Technology), rabbit anti-SPRED1 (1:1,000; ABCAm), mouse anti-VEGF (1:500; ABCAm), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:1,000; ABCAm). Next, anti-rabbit IgG (1:10,000; Aspen, Inc.) and anti-mouse IgG (1:10,000; Aspen, Inc.) were used as second antibodies at 1:2,000 for 30 min. Quantification of the protein bands were performed with AlphaEaseFC (Alpha Innotech, San Leandro, CA) software. Sample analyses were performed on day 9.
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