To induce HIF-1α upregulation, the cells were incubated with 100 μM CoCl2 in serum-free medium for 4 h. Then, the cells were treated with different formulations for 24 h, and lysed in RIPA buffer (Servicebio, China). After quantification of the isolated proteins using BCA protein quantification kit (Servicebio, China), equal amounts of proteins were loaded on 5% SDS-PAGE for separation, and then transferred to PVDF membranes for incubation with rabbit HIF-1α monoclonal antibody (1: 1000, ABclonel) for 3 h. Afterwards, secondary antibodies with horseradish peroxidase were incubated with the membrane for 30 min, and the proteins were visualized by a chemical luminescence kit (Servicebio, China). To measure the APBA1 expression, the cells were subjected with different treatments, and the total RNA was harvested by TRIzol reagent, and the relative level of ABCA1 mRNA was measured by RT-PCR (CFX-Connect, BIO-RAD, USA) according to the manufacturer’s instruction.
Bca protein quantification kit
Manufactured by Wuhan Servicebio Technology
Sourced in China
The BCA protein quantification kit is a reagent system used to determine the total protein concentration in a sample. It employs the bicinchoninic acid (BCA) method, which involves the reduction of copper ions by proteins in an alkaline medium, resulting in a purple-colored complex that can be measured colorimetrically.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Wuhan Servicebio Technology (1 mentions)
Cfx connect, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc pi apoptosis kit, by MultiSciences Biotech (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Wuhan Servicebio Technology (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Sds page gel preparation kit, by Wuhan Servicebio Technology (1 mentions)
Chemiluminescent substrate, by Bio-Rad (1 mentions)
Ripa lysate, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ab181020, by Abcam (1 mentions)
Ab108393, by Abcam (1 mentions)
Ab243894, by Abcam (1 mentions)
A0216, by Beyotime (1 mentions)
Gtx100027, by GeneTex (1 mentions)
A0208, by Beyotime (1 mentions)
4 protocols using bca protein quantification kit
1
Hypoxia-Inducible Factor-1α Modulation
2
Naringenin-Induced Apoptosis Pathway
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Naringenin (HPLC ≥ 98%) used in this study was acquired from Solarbio (IN0350, Solarbio Life Sciences, Beijing). Annexin V-FITC/PI apoptosis kit that was applied in flow cytometry was supplied by Multi Sciences Biotech (Hangzhou, China). The 5x protein loading buffer, BCA protein quantification kit, radio-immunoprecipitation assay (RIPA) buffer, SDS-PAGE gel preparation kit, 10x TBS with Tween-20 buffer, and phenylmethanesulfonyl fluoride (PMSF) all were bought from Servicebio Technology (Wuhan, China). Fetal bovine serum was purchased from Invitrogen (MA, USA). Polyvinylidene difluoride (PVDF) membrane and chemiluminescent HRP substrate were obtained from Millipore (Billerica, MA, USA). Sources and usage of other reagents are described in detail in the following instructions.
3
Western Blot Analysis of Protein Expression
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24 h after transfection, cells were harvested and lysed with 100 μl of RIPA lysate (Servicebio, Beijing, China) for 30 min on ice followed by centrifugation at 12000 rpm for 10 min at 4 °C, and subsequent collection of the supernatants. The concentration of protein was detected using the BCA Protein Quantification Kit (Servicebio, Beijing, China). About 30 μg of protein were separated by 10% sodium dodecyl sulphate polyacrylamide gels via electrophoresis (SDS-PAGE) (Bio-Rad) and electro-transferred to the PVDF membranes (Bio-Rad) (The PVDF membranes were activated with methanol prior to use). The membranes were then blocked in 3% BSA for 1 h. Subsequently, the membranes were incubated with the primary antibodies (TRAF3IP2, 1:3000; Cleaved-Caspase 3, 1:3000; Caspase 3, 1:3000; Bax, 1:3000; β-actin, 1:3000) overnight at 4 °C, and then washed three times in PBS-Tween followed by incubation in the appropriate secondary antibody (1:5000, Servicebio, Beijing, China) for 1 h at room temperature. After the membranes were washed three times with PBS-Tween, the chemiluminescent substrate (Biorad) was added to the membranes in order to detect proteins. Densitometric analysis was performed and protein levels were quantified using ImageJ software.
4
Quantifying Protein Expression in H1 and H9 Cells
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H1 and H9 cells were cultured normally and total protein was extracted with RIPA high-efficiency lysate. The protein concentration was measured according to the instructions of BCA Protein Quantification Kit (G2026-200T, Servicebio, China). A pipetting gun was used to accurately add proteins of the same mass into the compression gel and the voltage and current conditions were determined according to the molecular weight of different proteins. The protein was separated at 120 V voltage for some time and transferred to the PVDF membrane. The membrane was kept at 250mA constant for 90 min, sealed with sealing solution (P0023B-100mL, Beyotime, China), and incubated with antibody MIF (1:1000, sc-53915, Santa Cruz, USA), CD74 (1:1000, ab108393, Abcam, UK), CD44 (1:1000, Ab243894, Abcam, UK), CXCR2 (1:1000, 20634-1-AP, Proteintech, China), CXCR4 (1:1000, Ab181020, Abcam, UK), CXCR7 (1:800, GTX100027, GeneTex, USA) at 4°C overnight, rinsed with PBS for three times, and then incubated with horseradish peroxidase (HRP) -conjugated secondary antibody goat anti-rabbit IgG (H+L) (1:1000, A0208, Beyotime, China) or goat anti-mouse IgG (H+L) (1:1000, A0216, Beyotime, China) at room temperature for 2 h. A hypersensitive ECL chemiluminescence kit (P0018S, Beyotime, China) was used to detect protein bands, and the relative protein content of each group was analyzed by ImageJ software.
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