CCSP-RAGE TG and control mice were sacrificed and BALF was removed as described [27 (link)]. Supernatants were assayed for total protein with a bicinchoninic acid (BCA) total protein kit (Thermo Scientific). Total numbers of pelleted cells were counted with a hemocytometer and cells stained with a modified Wright-Giemsa stain (Diff-Quik; Baxter, McGaw Park, IL) were subjected to a blinded manual differential cell count in which 200 cells were counted per slide, and the percent of total cells was determined. Counting was performed in triplicate and the average was obtained.
Bca total protein kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA (Bicinchoninic Acid) total protein kit is a colorimetric assay used to determine the total protein concentration in a sample. It relies on the reduction of copper (II) ions to copper (I) ions by proteins in an alkaline medium, and the subsequent chelation of the copper (I) ions with bicinchoninic acid to produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Diff quik, by Baxter (2 mentions)
Rabbit anti bcl 2, by Abcam (1 mentions)
M per cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti bax, by Proteintech (1 mentions)
Rabbit anti jak2, by Cell Signaling Technology (1 mentions)
Rabbit anti stat3, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Nuclear extraction kit, by Signosis (1 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
4 protocols using bca total protein kit
1
BALF Analysis of CCSP-RAGE Mice
2
Western Blot Analysis of Cell Signaling Proteins
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The cells in each group were lysed in M-PER cell lysis buffer (Thermo Fisher Scientific, USA). The total protein of each group was collected and quantified measuring with a BCA total protein kit (Thermo Fisher Scientific, USA). Proteins of the same volume were separated by SDS-PAGE. Then, the proteins were transferred to the semidry membrane. Subsequently, membranes were blocked in 5% BSA for 1 hour at room temperature and incubated with β-actin (1 : 2500, Proteintech), rabbit anti-BAX (1 : 250, Proteintech), rabbit anti-Bcl-2 (1 : 1000, Abcam), rabbit anti-JAK2 (1 : 1000, Cell Signaling Technology), and rabbit anti-STAT3 (1 : 1000, Cell Signaling Technology) antibodies at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies labeled with horseradish peroxidase (HRP) for 2 hour. The membrane was exposed to the developing solution of electrogenerated chemiluminescence (ECL) (Advansta, CA, USA). The optical density of protein bands was analyzed by ImageJ image analysis software.
3
BALF Analysis in Pregnant Rats
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BALF was performed in treated and control pregnant rats (n = 10) as outlined previously with slight modifications [19 (link)]. Briefly, control and treated animal lungs were inflated and fixed with 4% PFA for lavage to procure BALF. This was specifically harvested through the installation and recovery of seven boluses of PBS with a syringe attached to a catheter for a total of 30 mL/kg. BALF samples were centrifuged for 10 min and supernatants were assayed for total protein using a bicinchoninic acid (BCA) total protein kit (Thermo Scientific). Pelleted cells were counted and stained with a modified Wright–Giemsa stain (Diff-Quik; Baxter, McGaw Park, IL, USA) for a blinded manual differential cell count in which 200 cells were counted per slide to determine the percent of total cells.
4
Profiling Transcription Factor Activation in Pseudomonas-Infected Cells
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The epithelial cells were infected with P. aeruginosa mutants, as described above. At 1 h post-infection, the nuclear extracts were obtained using a nuclear extraction kit (Signosis, SK-0001) following the manufacturer’s instructions. Triplicates for each condition were obtained by completing three independent experiments. The amount of protein in each extract was measured using both the NanoDrop Protein quantification instrument (Thermo Fisher Scientific) and using a bicinchoninic acid assay (BCA) total protein kit (Thermo Fisher Scientific, 23,225). Triplicates for each condition were combined and the activation level of 48 different transcription factors in each condition was measured using the Transcription Factor Activation Profiling Plate Array I (Signosis, FA1001) according to the manufacturer’s instructions. Briefly, 10 µg nuclear extracts were incubated with biotin-labeled probes encoding TF DNA-binding site consensus sequences from 30 min at room temperature. The unbound probes were washed away using the provided isolation column. The remaining probes were eluted and hybridized to complementary sequences in provided 96-well hybridization plate at 42 °C overnight. The plates were washed, blocked, and the luminescence signal measured on Synergy™ HTX Multi-Mode Microplate Reader (BioTek). The values were normalized to (ER Estrogen Receptor).
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