Lipofectamine™ 3000 transfection reagent was obtained from Invitrogen (Carlsbad, USA). Cell counting kit-8 (CCK-8) was provided by Dojindo (Kumamoto, Japan). anti-ERK1ERK2 (phospho T202/Y204, SP327), anti-c-Myc (phospho S62) antibody, anti-MNK1 (phospho T385, EPR2370), anti-ERK1+ ERK2, anti-MNK1 and anti-c-Myc antibodies were obtained from Abcam (London, United Kingdom). PTPRF/LAR (E6W4X) rabbit mAb was obtained from Cell Signaling Technology (Massachusetts, United States). BCA protein concentration assay kit and 5× protein loading buffer were provided by Beijing Solarbio life sciences(Beijing, China). NucleoZol was obtained from Gene Co., Ltd (Hongkong, China). ECL luminescence reagent was obtained from Thermo Scientific (Waltham, USA). qPCR Mix was provided by PROMEGA (Madison, MA, United States).
Bca protein concentration assay kit
Manufactured by Solarbio
Sourced in China, United States
The BCA protein concentration assay kit is a colorimetric assay used to determine the total protein concentration in a sample. It is based on the reduction of copper ions by proteins in an alkaline medium, which results in the formation of a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Pvdf membrane, by Solarbio (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Primescript 2 rtase, by Takara Bio (2 mentions)
Ifn γ, by Bioswamp (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Anti c myc antibody, by Abcam (1 mentions)
Protein loading buffer, by Solarbio (1 mentions)
Ecl luminescence reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Anti erk1 erk2, by Abcam (1 mentions)
Opti mem, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sybr fast qpcr master mix, by Roche (1 mentions)
40 protocols using bca protein concentration assay kit
1
Lipofectamine 3000 Transfection and Analysis
2
Apoptosis Regulation in Leukemia Cells
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Human promyelocytic leukemia cells (HL-60) and acute lymphatic leukemia cells (CCRF-CEM) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. DAC (A119533) was purchased from Aladdin (China). Lipofectamine® 2000 (11668-027) was purchased from Invitrogen (China). Opti-MEM (M5650) was purchased from Sigma (USA). The dual luciferase reporter gene assay kit (RG027) was purchased from Beyotime (China). The cell counting kit 8 (CCK8) (C1706) was purchased from Bioswamp (China). The SYBR FAST qPCR Master Mix (KM4101) was purchased from KAPA Biosystems (USA). The oligo DT18/miR-135a-5p RT Primer (3806), recombinant RNase inhibitor (2313A), and PrimeScript II RTase (2690A) were purchased from TAKARA (Japan). BCA protein concentration assay kit (PC0020) was purchased from Solarbio (China), DAB (DA1010) was purchased from Solarbio (China), and all antibodies (anti-Bad PAB32756; anti-Bcl-2 PAB30599; anti-cleaved-caspase-3 MAB37300; anti-GAPDH PAB36269; goat anti-rabbit IgG SAB43714) were purchased from Bioswamp (China).
3
Protein Extraction and Western Blot Analysis
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Cell lysis was performed using the RIPA (including 1% PMSF) protein extraction solution (Solarbio, Beijing, China). The protein concentrations were measured using the BCA protein concentration assay kit (Solarbio, Beijing, China). SDS–polyacrylamide gel electrophoresis (PAGE) was conducted to separate the extracted proteins with different molecular weights. The target proteins were immediately transferred to nitrocellulose membranes, which were then incubated overnight at 4℃ with antibodies against β-actin (42 kDa; ZSGB-BIO, Beijing, China), β-Tubulin (55 kDa; ZSGB-BIO, Beijing, China), GPR40 (40 kDa; Abcam, Cambridge, USA), GPR120 (40 kDa; Abcam, Cambridge, USA), KLF7 (25 kDa; Abcam, Cambridge, USA), CCL2 (40 kDa; Abcam, Cambridge, USA), CCR2 (35 kDa; San Ying Biotechnology, Wuhan, China), Ki67 (384 kDa; Abcam, Cambridge, USA), and MMP2 (72 kDa; Abcam, Cambridge, USA). Afterward, the membranes were incubated with the secondary antibody at room temperature for 2 h. It needs to be explained that, in order to reduce the mutual interference of polyclonal antibody incubation among multiple antibodies, we have tailored according to MW markers in this study, and each membrane containing different target proteins is incubated separately with corresponding antibodies.The protein bands were detected based on chemiluminescence (ThermoScientific, Waltham, America).
4
Protein Analysis of Retinal Tissues
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The total protein was isolated from retinal tissues using RIPA reagent (Solarbio) containing PMSF (Solarbio). Afterwards, the protein concentration was quantified by a BCA protein
concentration assay kit (Solarbio), and electrically separated by 10% sodium dodecyl sulfate-polyacrylamide gel. Protein in the target region was transferred to the PVDF membranes
(Millipore, Billerica, MA, USA), which were then blocked in 5% skimmed milk (Sangon) for 1 h at room temperature. The blocked PVDF membranes were incubated in primary antibodies overnight at
4°C and then in goat anti-rabbit/mouse HRP-conjugated secondary antibody 37°C for 1 h. Protein bands were visualized via chemiluminescent (ECL) (Solarbio, China) and quantified by
Gel-Pro-Analyzer software. Antibodies used here were shown inSupplementary Table 1 .
concentration assay kit (Solarbio), and electrically separated by 10% sodium dodecyl sulfate-polyacrylamide gel. Protein in the target region was transferred to the PVDF membranes
(Millipore, Billerica, MA, USA), which were then blocked in 5% skimmed milk (Sangon) for 1 h at room temperature. The blocked PVDF membranes were incubated in primary antibodies overnight at
4°C and then in goat anti-rabbit/mouse HRP-conjugated secondary antibody 37°C for 1 h. Protein bands were visualized via chemiluminescent (ECL) (Solarbio, China) and quantified by
Gel-Pro-Analyzer software. Antibodies used here were shown in
5
Protein Quantification and Western Blot Analysis
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Proteins were extracted from lung tissues and quantified using the BCA protein concentration assay kit (Solarbio, China). Equal amounts of protein were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBST (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5% Tween-20) for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used according to the manufacturer’s protocol: anti-MMP9 (ab76003), anti-TIMP1 (ab109125), and anti-Caspase3 (ab32351) from Abcam (Cambridge, Cambridgeshire, UK); anti-Bax (#2772) from Cell Signaling Technology (Danvers, MA, USA); anti-GAPDH from Proteintech (Chicago, IL,USA); anti-MMP12 (bs-1854R) from Bioss (Beijing, China), Anti-Caspase8(WL02434), anti-Bcl2 (WL01556), and anti-Survivin (WL01684) from Wanleibio (Shenyang, Liaoning, China). The membranes were washed in TBST and incubated with secondary antibodies for 1.5 h. After extensive washing, the membranes were visualized using enhanced chemiluminescence reagent. Final images were analyzed using ImageJ software.
6
ATP Quantification in Protein Samples
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The ATP production was measured according to the kit (S0026, Beyotime, Shanghai, China) instructions; the RLU value was measured and calculated according to the standard curve to obtain the ATP concentration of the sample using a Luminometer (Biotek, Germany). The protein concentration in the sample was determined using the BCA Protein Concentration Assay Kit (PC0020-500, Solarbio Beijing, China). The concentration of ATP was subsequently converted to the form of nmol/mg protein.
7
Exosome-mediated Regulation of B Cell Apoptosis
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hucMSCs were plated in six-multiwell flat bottom culture plates (NEST, China) at 5 × 104 cells/well density and cultured in basal medium supplemented with FBS (10%). After 6 hr for cell adhesion, the medium was aspirated and replaced with fresh PBMCs at 5 × 105 cells/well, corresponding to a ratio of hucMSCs/PBMC 1 : 10. To assess apoptosis, PBMCs were collected after coculture using Annexin V-FITC/PI apoptosis kit (MULTI Sciences, China) according to the manufacturer's instructions, centrifuged at 1,500 rpm for 5 min, washed with PBS, and resuspended in 500 μL 1 × binding buffer. B cells were sorted by sequentially adding 5 μL CD19 (APC, Biolegend, USA), then incubated with FITC and PI for 15 min at room temperature and then analyzed by flow cytometry (Agilent NovoCyte, Model number: D2040, China).
The concentration of purified hucMSCs-Exo was measured via BCA protein concentration Assay Kit (Solarbio, China). To select the optimal concentration hucMSCs-Exo for the subsequent experiments, a series of concentrations of hucMSCs-Exo, including 20, 50, 100, and 200 μg/mL, was prepared. The optimal concentration was determined according to the minimum concentration that could promote cell apoptosis.
The concentration of purified hucMSCs-Exo was measured via BCA protein concentration Assay Kit (Solarbio, China). To select the optimal concentration hucMSCs-Exo for the subsequent experiments, a series of concentrations of hucMSCs-Exo, including 20, 50, 100, and 200 μg/mL, was prepared. The optimal concentration was determined according to the minimum concentration that could promote cell apoptosis.
8
Western Blotting of Angiogenic Markers
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Western blotting was carried out as previously described.23 (link) Protein of each sample was quantified by BCA protein concentration assay kit (Cat# PC0020, Solarbio, Beijing, China). The loading quantity was 30 μg of each sample, and proteins were distinguished by SDS‐PAGE (sodium dodecyl sulphate‐polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene fluoride) membrane for blotting. For CD34, the antibody for Western blot was the same as for immunohistochemistry, and dilution was 1:1000. The other antibodies were purchased from Abcam (Shanghai, China), namely, CD31 (Cat# ab28364, 1:500), CDH5 (Cat# ab33168, 1:1000) and VEGFA (Cat# ab1316, 1:500).
9
Quantitative Analysis of PI3K-Akt Signaling
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We seeded HeLa cells in 6-well plates at a cell density of 3 × 105 cells/mL, then we treated the cells with 160 μg/mL of FFGL at different time points. Subsequently, the HeLa cells were lysed in radioimmunoprecipitation assay buffer for 30 min on ice. After extracting the total protein content, we quantified it using a bicinchoninic acid (BCA) Protein Concentration Assay Kit (Solarbio, Beijing, China) and separated the proteins via 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The separated proteins were then transferred to a polyvinylidene fluoride membrane. Next, they were blocked with 5% skim milk for 45 min and incubated overnight with the following primary antibodies: anti-PI3K (1:1000, CST, Danvers, MA, USA) and anti-Akt (1:1000, CST, USA). Subsequently, the membrane was washed thrice with tris-buffered saline (TBS) containing 0.1% Tween-20 and incubated with a secondary antibody (1:5000, CST, USA) for 1 h. The membrane was exposed to an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) and then imaged using an infrared laser scanning instrument (Analytik Jena, Jena, Germany). Protein bands were scanned and analyzed using ImageJ software for optical densitometry for the semi-quantitative determination of the protein expression levels. The experiment was repeated three times.
10
Quantifying Protein Expression in Rat Blood
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Total protein was extracted from rat blood samples using RIPA protein lysate (Solarbio, Beijing, China). Protein content was quantified using the BCA Protein Concentration Assay Kit (Solarbio). The proteins were separated by 8% SDS-PAGE and transferred to PVDF membranes, which were closed with 5% skimmed milk at ambient temperature for 2 h. Next, the membranes were subjected to cultivation with primary antibodies against JAK2 (1:500), p-JAK2 (1:1000), STAT3 (1:500), p-STAT3 (1:1000) and β-actin (1:1000) (all from Cell Signaling Technology, Inc., USA) overnight at 4 °C, followed by 1 h-cultivation with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody at 37 °C. Protein bands were tested using an ultra-sensitive enhanced chemiluminescence kit (Beyotime, Shanghai, China). The grayscale values of each protein band were analyzed using Image-Pro Plus (version 6.0, Media Cybernetics, Inc.).
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