Retina tissues were freshly dissected 1 day, 3 days, and 7 days after AOH injury and then homogenized in radioimmunoprecipitation assay buffer containing a protease and phosphatase inhibitors cocktail. Protein concentration was measured with the BCA Protein Assay Kit (BL521A; Biosharp, Hefei, China) according to the manufacturer's instructions. Protein samples (20 µg) from each sample were loaded into 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat powdered milk for 1 hour at room temperature (RT) and then incubated with primary antibodies (Table ). Then the membranes were washed three times and incubated with corresponding horseradish peroxidase–conjugated goat anti-mouse (1:5000, A21010; Abbkine, Wuhan, China) or anti-rabbit (1:5000, A21020; Abbkine) secondary antibodies for 1 hour at RT. After washing, proteins were detected with enhanced chemiluminescence reagent (FD8030; FDbio, Hangzhou, China) using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). The band intensity was quantified by Image Lab 6.0 software (Bio-Rad).
Fd8030
Manufactured by Fdbio
Sourced in China
The FD8030 is a laboratory equipment designed for freeze-drying applications. It is capable of reducing the moisture content of samples through a process of sublimation. The device operates at controlled temperatures and pressure conditions to facilitate the drying process. The FD8030 is suitable for use in various research and industrial settings where sample preservation is required.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay reagent, by Beyotime (2 mentions)
Fd1001, by Fdbio (2 mentions)
Fdr007, by Fdbio (2 mentions)
Fdm007, by Fdbio (2 mentions)
Image lab 6, by Bio-Rad (1 mentions)
A21010, by Abbkine (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
A21020, by Abbkine (1 mentions)
Bca protein assay kit, by Biosharp (1 mentions)
Mouse anti β actin antibody a5441, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Radio immunoprecipitation assay lysis buffer, by Meilun (1 mentions)
Bcl 2 12789 1 ap, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Fd002, by Fdbio (1 mentions)
Ma0151, by Meilun (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
6 protocols using fd8030
1
Retinal Protein Expression Dynamics After AOH
2
Western Blot Analysis of PGC1β, CPT1α, and CPT1β
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Total protein was isolated from the liver in a lysis buffer for 30 min at 4°C. Protein concentration was measured using BCA Protein Assay Reagent (P0011, Beyotime Biotechnology, China). The proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat dry milk in PBS with 0.02% (v/v) Tween-20, and incubated with primary antibodies at 4°C overnight. The membrane was washed and incubated for 1 h at room temperature with a peroxidase-labeled secondary antibody. After washing, protein bands were visualized by electrochemiluminescence (FD8030, FDBio Science, China). Anti-Pgc1β (A17089), Cpt1α (A5307), and Cpt1β (A6796) were obtained from ABclonal (Wuhan, China). Mouse anti-β-actin antibody (A5441) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Protein Expression Analysis in Cell Lysates
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The cells and tissues were lysed on ice with Radio-Immunoprecipitation assay lysis buffer (MA0151, MeilunBio, China) and a mixture of protease inhibitors (FD1001, FDBio, Hang, China). Protein expression was detected in sulfate polyacrylamide gel electrophoresis. Electroblot of the protein was onto a polyvinylidene fluoride membrane (Amersham Bioscience, NJ). The sealing of the membrane was required with Tris buffered saline and 5% skim milk powder, with incubation of primary antibodies and Horseradish peroxidase-conjugated secondary antibodies (FDM007 and FDR007, FDBIO). To this end, protein measurements were implemented with an enhanced chemiluminescence kit (FD8030, FDBIO). The primary antibodies were as follows: cleaved caspase-3 (9664, Cell Signaling Technology), Bax (2772, Cell Signaling Technology), Bcl-2 (12789-1-AP, ProteinTech), NAMPT (11776-1-AP, Proteintech).
4
Western Blot Analysis of Extracellular Matrix Proteins
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (MA0151, MeilunBio, China) with a protease inhibitor cocktail (FD1001, FDbio, Hangzhou, China) for 20 min on ice. After 20 min, 5× loading buffer (FD002, FDbio) was added to the lysis buffer. Protein expression was detected by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 15 min and 120 V for 90 min. The proteins were then transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane (Amersham Bioscience, NJ) with transfer buffer at 300 mA for 100 min. The PVDF membrane was blocked with Tris-buffered saline and Tween 20 (TBST) containing 5% dissolved skim milk powder at room temperature for 1 h. Next, the PVDF membrane was incubated with primary antibodies overnight at 4°C. Then, the membrane was rinsed three times with TBST for 10 min. A horseradish peroxidase (HRP)-conjugated secondary antibody (FDM007 and FDR007, FDbio) was then added to the membrane and incubated for 1 h at room temperature. Finally, protein levels were detected using an enhanced chemiluminescence kit (FD8030, FDbio). Abcam (Cambridge, United Kingdom) provided anti-β-actin, anti-MMP3, anti-MMP13, anti-COL2A1, anti-aggrecan, and anti-ADAMTS4 antibodies. Primary antibody dilution buffer was provided by MeilunBio (Dalian, China).
5
Asiaticoside Modulates LPS-Induced Inflammation
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RAW264.7 cells were treated with LPS (100 μg/ml) or LPS (100 μg/ml) + asiaticoside (100 μM) for 48 h. Next, cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) for 20 min at 4°C to extract total protein. Equal amounts (40 µg) of protein lysates were resolved by SDS-PAGE on 10% gels, and then transferred to a PVDF membrane. After blocking with 5% nonfat dry milk in PBS solution containing 0.02% (v/v) Tween-20, membranes were incubated with the primary antibodies (all 1:1000) at 4°C overnight. On the next day, membranes were washed and incubated with a peroxidase-labeled secondary antibody (1:5000) at room temperature for 1 hour. After washing, the protein bands were visualized by electrochemiluminescence (FD8030, FDBio Science, China).
6
Protein Isolation and Western Blotting
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Total protein was isolated from eWAT in lysis buffer for 30 min at 4°C. Protein concentrations were measured using BCA Protein Assay Reagent (P0011, Beyotime Biotechnology, China). Proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat dry milk in PBS with 0.02% (v/v) Tween-20, and incubated with the primary antibodies at 4°C overnight. Next, the membrane was washed and incubated for 1 h at room temperature with a peroxidase-labeled secondary antibody. After washing, protein bands were visualized by electrochemiluminescence (FD8030, FDBio Science, China). The anti-mitochondrial oxidative complex cocktail antibody (ab110413, 1:200 dilution) and anti-hnRNPA1 antibody (sc365486) were obtained from Abcam (Cambridge, UK) and Santa Cruz (USA), respectively. The mouse anti-b-actin antibody (A5441) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
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