Mix 1 µg antibody with 5 × SDS-PAGE Protein Loading Buffer (YEASEN, # 20315ES05) and boil. SDS-PAGE and Coomassie Brilliant Blue staining were performed. The light chain and heavy chain of the antibody were cut off, digested with trypsin, and sent to BGI company for mass spectrometry analysis by MicrOTOF-QII equipment (BrukerDaltonics, Billerica, MA, USA).
Sds page protein loading buffer
Manufactured by Yeasen
Sourced in China
SDS-PAGE Protein Loading Buffer is a solution used in the preparation of protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. It is designed to denature and solubilize proteins, enabling their separation by molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Microtof q 2 equipment, by Bruker (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Sds page gel preparation kit, by Yeasen (1 mentions)
Ripa lysis buffer, by Yeasen (1 mentions)
Instab protease cocktail, by Yeasen (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
6 protocols using sds page protein loading buffer
1
Antibody Characterization by Mass Spectrometry
2
Western Blot Analysis of Fetal Cortex
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Fetal cerebral cortex from both Ctr and Sevo groups at E14.5 was lysed in the lysis buffer and centrifuged at 13 000 rpm for 30 minutes at 4°C. The supernatant was collected and mixed with SDS‐PAGE Protein Loading Buffer (Yeasen), then boiled at 95°C and separated by 10% SDS‐polyacrylamide gel electrophoresis (PAGE). The proteins were transferred onto polyvinylidene difluoride membranes. The membranes were incubated with 5% non‐fat dry milk for 2 hrs at room temperature and primary antibody at 4°C overnight. After washed by TBST three times, the membranes were incubated with secondary antibody for 1 hr at room temperature, followed by washing with TBST three times. Blots were detected by ECL luminescence reagents (BBI) and imaged using ChemiDoc Imaging System (Bio‐rad). The bands were quantified by densitometry (ImageJ) to determine the expression of the protein. The ratio of band density of NICD over GAPDH was calculated.
3
Acetylation of N-Myc by p300
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Recombinant glutathione-S-transferase (GST)-N-Myc protein (catalog no.: ABIN1311728; Antibodies-online) was incubated with recombinant p300 protein (catalog no.: 81158; Active Motif) with or without the p300 inhibitor CPI-637 (catalog no.: S8190; Selleck) in 50 mM Tris buffer (pH 8.0) containing 2% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and acetyl-CoA for 1 h at 30 °C (23 (link)). Products were then prepared for MS analysis or boiled with SDS-PAGE protein loading buffer (catalog no.: 20315ES05; Yeasen) for Western blot analysis.
4
ACE2 Expression Analysis in Mice Organs
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Vital organs removed from normal mice were crushed with a mortar using liquid nitrogen under ice cold conditions and lysed with RIPA lysis buffer (YEASEN, #20101ES60) and InStab™ Protease Cocktail, EDTA-free, mini, tablet-form (YEASEN, #20123ES10). After centrifugation at 14,000 r/min at 4 °C for 20 min, supernatants were collected and protein concentration was determined using the Pierce™ BCA protein assay. All samples were heated for 10 min at 95 °C in 5× SDS-PAGE Protein Loading Buffer (YEASEN, #20315ES05) before loading. Cell lysate aliquots were separated in 10% SDS-PAGE (SDS-PAGE Gel Preparation Kit, YEASEN, #20328ES50). Proteins were electro-blotted onto polyvinylidene fluoride membranes (Millipore) and blocked by incubation with TBST (TBS and 0.2% Tween-20) containing 5% non-fat dry milk for 1 h at room temperature. After blocking, membranes were washed in TBST three times for 5 min and then probed with the Recombinant Anti-ACE2 Momoclonal Antibody (Abcam, #ab108252) overnight at 4 °C. Subsequently, the membranes were washed with TBST and incubated for 1 h at room temperature with goat-anti rabbit-HRP (Servicebio, #GB23303) in TBST. Finally, membranes were washed three times and enhanced with Clarity™ Western ECL substrate (YEASEN, #36208ES60), followed by imaging with Alliance Micro Q9 (UVITEC).
5
Western Blot Analysis of A2780 and ID8 Cells
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A2780 cells (6 × 105 cells per well) and ID8 cells (3 × 105 cells per well) were seeded into six-well plates. Cell lysates were mixed with 5 × SDS-PAGE Protein Loading Buffer (Yeason, Shanghai, China) and boiled at 95 °C. Protein samples were loaded onto PAGE gels and transferred to PVDF. After blocking with 5% non-fat milk dissolving in PBST for 1.5 h at RT, membranes were incubated with primary antibody overnight at 4 °C. The next day, membranes were washed and incubated with horse radish peroxidase (HRP)–conjugated secondary antibodies for 1.5 h at RT. Bands were detected using Syngene Bio Imaging (Synoptics, Cambridge, UK).
6
Plasmid Transfection and Western Blotting Protocol
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The DF-1 cells were plated in 12-well plates at 1 × 106/1 mL and then transfected with empty plasmids or various expression plasmids. After 24 h, the transfected cells were washed twice with phosphate buffer saline (PBS) (Gibco) and then lysed with a cell lysis buffer (Beyotime, Shanghai, China) containing an InStab™ protease cocktail (Yeasen, Shanghai, China) and phenylmethylsulfonyl fluoride (PMSF) (Yeasen). The cell lysates were centrifuged at 13 000 rpm for 15 min to obtain the supernatant, and a 5 × SDS-PAGE protein loading buffer (Yeasen) was added. The lysates were then boiled for 10 min. The proteins isolated from the cell lysates were separated via SDS-PAGE and analyzed using a Western blot. Images were obtained using the Tanon 5200 imaging system (Tanon, Shanghai, China), as described in our previous study [42 (link)].
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