Cells or tissues were lysed using RIPA buffer. BCA Protein Assay Kit II (Biovision, USA) was used to measure protein concentration. The antibodies include cyclin A1 (Proteintech, USA), cyclin B1 (Proteintech, USA), CDK2 (Proteintech, USA), PDPK1 (Proteintech, USA), and β-actin (Proteintech, USA). HRP-labeled goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Cell Signaling Technology. The process of western blotting was referred to in the previous article [17 (link)].
Bca protein assay kit 2
Manufactured by Abcam
Sourced in United States
The BCA Protein Assay Kit II is a colorimetric assay used to determine the total protein concentration in a sample. The kit utilizes the well-established bicinchoninic acid (BCA) method, which combines the reduction of Cu2+ to Cu+ by proteins in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) using a reagent containing bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
β actin, by Proteintech (1 mentions)
Cyclin a1, by Proteintech (1 mentions)
Pdpk1, by Proteintech (1 mentions)
Cyclin b1, by Proteintech (1 mentions)
Dy400, by R&D Systems (1 mentions)
Dy1679, by R&D Systems (1 mentions)
Dy406, by R&D Systems (1 mentions)
Dy410, by R&D Systems (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Vortex genie 2 mixer, by Scientific Industries (1 mentions)
Las 4000, by Cytiva (1 mentions)
6 protocols using bca protein assay kit 2
1
Protein Extraction and Western Blotting
2
Hemolymph Protein Quantification using BCA
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Protein was measured using a BCA Protein Assay Kit II (BioVision Inc., Milpitas, CA) by adding 25μL of the hemolymph solution to 200μL of the BCA working reagent in each of the Costar® 96 well flat bottom plate wells. The plate was covered and incubated at 37°C for 30 minutes. After the incubation the absorbance was measured at 562nm. A standard curve using the provided standards was utilized to determine protein concentration (μg/mL). Once protein concentrations were known, phenoloxidase units were expressed as phenoloxidase units per mg of protein.
3
Cytokine Profiling of Tumor Tissues
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Tumor tissue samples (100 mg) were treated with 0.5% TRITON-X100 in PBS and homogenized. After centrifugation, supernatants were collected and frozen. The following ELISA kits were used according to the manufacturer’s instructions: DY400, DY410, DY1679, DY406 (R&D Systems, Minneapolis, MN, USA), 900-M47, 900-T53 (Peprotech, Rocky Hill, NJ, USA). The cytokine concentration was normalized to the total protein concentration, and measured using a BCA Protein Assay Kit II (K813-2500, BioVision, Milpitas, CA, USA).
4
Protein Extraction and Western Blot Analysis
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Protein extracts were obtained from gastrocnemius muscles using lysis buffer (100 mM Tris-HCl, pH 6.8, 2.0% SDS, 20% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol, 100 mM NaF and 1 mM Na3VO4) and the protein concentration of lysates was determined using a BCA Protein Assay Kit II (BioVision, Milpitas, CA, USA). Primary and secondary antibodies used in this study are listed in Key Resources Table . Target proteins were detected using an Odyssey Infrared Imaging System (LI-COR Biosciences) as previously described (Son et al., 2019b ). All uncropped images are listed in Data S1 .
5
Isolation and Quantification of Ileal Epithelial Proteins
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Upon collection, tissues were frozen immediately with liquid nitrogen and stored at -80˚C. For protein extraction, isolated ileal epithelium was subjected to ice-cold RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing proteinase inhibitor cocktails (Millipore) and homogenized by beads-beating using Vortex-Genie 2 mixer (Scientific Industries, Inc.) for 30 min at 4˚C. After centrifugation, the supernatant was collected for immunoblot analysis. The total protein concentration was determined using the BCA Protein Assay Kit II (BioVision) and the measurements were detected using the Epoch microplate spectrophotometer (BioTek). For each sample, 10 μg of extracted protein was used for electrophoresis. The primary antibody against DUSP6 (Dilution: 1:3000, Cat# ab76310, RRID: AB_1523517, Abcam) and Actin (Dilution: 1:5000, Cat# MAB1501, RRID:AB_2223041, Sigma-Aldrich) were used in this study. Protein marker (PM2510, SMOBIO Technology, Inc.) was employed to determine the size of target proteins. The luminescence intensity was detected by the ImageQuant LAS 4000 mini-imaging system (GE Healthcare Life Sciences). Quantification of proteins on immunoblots was performed with ImageJ software (RRID:SCR_003070, NIH). Original uncropped blots were provided in the Supplementary Information. All blots were processed in parallel and derive from the same experiment.
6
Protein Extraction and Western Blot Analysis
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