Forty-eight hours after transfection, total protein was extracted in an ice bath at 4°C using the ProteoPrep Sample Extraction Kit (Sigma-Aldrich, St Louis, MO, USA). Protein levels were quantified by a nucleic acid/protein analyzer. Forty µg of protein was separated by 12% sodium dodecyl polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with bovine serum albumin (Solarbio, Beijing, China) for 2 hours at room temperature, and incubated for 2 hours at 37°C. Nitrocellulose membranes were washed with PBS-Tween, incubated for 2 hours at 37°C, and incubated with a chemiluminescence reagent from the DAB Substrate Kit (US Everbright Inc., Suzhou, China) for ∼2 minutes. Proteins were visualized using a luminescence imager, and the absorbance of each band was measured using ImageProPlus software (Media Cybernetics Inc., Shanghai, China). Three replicates were used for each group. The relative expression of the target protein was defined as the ratio of the absorbance of the target band to that of the internal control band.
Proteoprep sample extraction kit
Manufactured by Merck Group
Sourced in United States
The ProteoPrep Sample Extraction Kit is a laboratory equipment product designed for the extraction and purification of proteins from various sample types. The kit contains the necessary components and reagents to facilitate the efficient isolation of proteins, which is a crucial step in many biological and biochemical analyses.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (1 mentions)
Sds page 10 gel, by Bio-Rad (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Rabbit anti p62, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using proteoprep sample extraction kit
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Autophagy and Inflammation Markers
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Total protein was extracted from tissues and NSCLC cells using ProteoPrep Sample Extraction kit (Sigma-Aldrich; Merck KGaA). The QuantiPro™ BCA Assay kit (cat. no. QPBCA; Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Protein samples (30 µg) were separated via SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk in TBS with 0.05% Tween-20 for 1 h at room temperature. After the membranes were incubated with primary antibodies at 4˚C overnight, secondary antibodies were added and incubated with the membranes at room temperature for 1 h. The bands were visualized with an ECL Western Blotting substrate kit (cat. no. K820; BioVision, Inc.) and the protein expression was quantified using ImageJ v1.8.0 software (National Institutes of Health) with GAPDH as the loading control. The primary antibodies used included: Mouse anti-IL-1β (1:1,000; cat. no. 12242; Cell Signaling Technology, Inc.), rabbit anti-LC3B (1:1,000; cat. no. ab51520; Abcam), rabbit anti-P62 (1:10,000; cat. no. ab109012; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). The corresponding secondary antibodies included: Anti-mouse IgG HRP-conjugated antibody (1;2,000; cat. no. 7076; Cell Signaling Technology, Inc.) and goat anti-rabbit IgG HRP-conjugated antibody (1:20,000; cat. no. ab205718; Abcam).
3
Enzyme-Linked Immunosorbent Assay for H. pylori
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Overnight cultures of bacteria were collected by centrifugation (5,000×g, 10 min, 4°C), washed three times with PBS, and repelleted at 10,000×g for 2 min. Total proteins were extracted by using the ProteoPrep® Sample Extraction kit (Sigma-Aldrich Corporation, St. Louis, MO, USA) according to the manufacturer’s instructions. The protein concentration was measured by the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Microplates were pretreated with 2.5% glutaraldehyde, and each ELISA plate was coated with 100 µl H. pylori at a concentration of 1 mg/mL in PBS and incubated overnight at 4°C. The coated wells were blocked with PBS containing 2.5% bovine serum albumin (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 2 h at 37°C. A 100 µL milk sample, which was diluted 1:200, 1:400, 1:800, and 1:1,200 with PBS containing 0.05% Tween 20 was dispensed to the microplate. Horseradish-peroxidase-conjugated goat anti-bovine IgG antibody (Southern Biotechnology Associates Inc, Birmingham, AL, USA) which was diluted 1:8,000 and tetramethylbenzidine peroxide substrate systems were used as the secondary antibody and the substrate, respectively. Absorbance at 450 nm (A450) was measured after the reaction was terminated with 1 M H2SO4. The maximal milk dilution giving an A450 of 0.5 was expressed as the antibody titer.
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