Ovarian tissue samples were homogenized in PBS with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ, USA) on ice and sonicated. Protein concentrations were determined by BCA protein assay (TIANGEN BIOTECH, Beijing, China). The protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (BioTrace™ NT, Port Washington, NY, USA). The membranes were blocked in 5% nonfat dry milk in tris-buffered-saline with tween 20 (TBST) for 1 h and incubated with a primary antibody against SIRT1, SIRT6, and FOXO3a (1:200 dilution, Santa Cruz Biotechnology, CA, USA), NRF1 (1:400 dilution, Santa Cruz Biotechnology, CA, USA), p53 (1:600 dilution, Santa Cruz Biotechnology, CA, USA) or β-action (1:1,000 dilution, Santa Cruz Biotechnology, CA, USA) over-night at 4°C, followed by the incubation with a secondary (horseradish peroxidase-conjugated) anti-rabbit or anti-mouse antibody (1:5,000 diluted) at room temperature for 1 h. Bands were visualized with a chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). Band intensities were analyzed using the Quantity One software (Bio-Rad Laboratories Pty. Ltd., Hercules, CA, USA). β-actin was used as a loading control.
Bca protein assay
Manufactured by Tiangen Biotech
Sourced in China
The BCA protein assay is a colorimetric detection and quantification method used to determine the total protein concentration in a sample. The assay utilizes the bicinchoninic acid (BCA) reagent, which reacts with the peptide bonds in proteins, producing a purple-colored complex that can be measured using a spectrophotometer.
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Lab products found in correlation
Teflon glass homogenizer, by Thomas Scientific (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Nrf 1, by Santa Cruz Biotechnology (1 mentions)
β action, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab2077, by Abcam (1 mentions)
Anti aβ 6e10, by Fortrea (1 mentions)
Pdvf membrane, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using bca protein assay
1
Western Blot Analysis of Ovarian Proteins
2
Hippocampal Protein Extraction and Analysis
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Protein extraction from the hippocampus was performed as previously described [19 (link)]. Protein concentration was determined using the BCA protein assay (Tiangen Biotech Co. Ltd., Beijing, China). 10 µg of protein from each sample was separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) for immunoblotting. The following primary antibodies were used: anti-Aβ (6E10, 1:1000; Covance, Emeryville, CA, USA), anti-BACE1 (1:500, ab2077; Abcam, Cambridge, MA, USA), and anti-β-Actin (1:500, ab2077; Abcam). Band intensities were semi-quantified using ImageJ ver. 1.6 (http://imagej.nih.gov/ij/ ) software.
3
Protein Extraction and Western Blot Analysis
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Cells were lysed in lysis buffer supplemented with phenylmethanesulfonyl fluoride (PMSF) to extract proteins. The protein concentration was detected by using the BCA protein assay (Tiangen, China). 30 μg proteins per lane were separated by 10% SDS-PAGE and transferred to PDVF membrane (Millipore, Billerica, USA). The PVDF membrane was placed in 5% skimmed milk and blocked, and the primary (PCNA (ab18197, 1 : 1000), MMP-3 (ab53015, 1 : 1000), Bax (ab53154, 1 : 1000), LC3 II/I (ab48394, 1 : 500), P62, Beclin1 (ab91526, 1 : 500), AMPK (ab3760, 1 : 1000), p-AMPK (ab109402, 1 : 1000), Mtor (ab2732, 1 : 2000), p-mTOR (ab84400, 1 : 400)) and secondary antibodies (ab205718, 1 : 2000) were incubated (all primary antibodies were purchased from Abcam, Cambridge, MA) and tested with a ChemiDoc XRS imaging system.
4
Purification and Analysis of CidA-CidB Complexes
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E. coli BL21(DE3) expressing either His-tagged CidA mutants or GST-tagged CidBND1-ND2 variants were harvested by centrifugation and resuspended separately in a buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM PMSF and 5% glycerol. The expression levels of CidA variants were approximated by the BCA Protein Assay (Tiangen Biotech (Beijing) Co., Ltd., China). Equal amount of CidA variants were mixed with a large and fixed volume of CidBND1-ND2 variants to ensure that CidBND1-ND2 variants were in excess to CidA. The mixture was co-lysed by sonication. Cleared lysates were incubated with 50 μL Ni-NTA resin at 4 °C for 1 h. The resin was washed eight times with 800 μL buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.01% Tween-20, 50 mM imidazole each time. Proteins were eluted by adding 3 resin volumes of 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 300 mM imidazole. The samples were analyzed using the SDS-PAGE and Coomassie stain.
5
Western Blot Analysis of Cell Lysates
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Cells were lysed in RIPA buffer containing protease inhibitors on ice for 30 min, then centrifuged for 10 min at 10,000 g. Supernatants were taken for protein measurement with the BCA protein assay (Tiangen). Samples were boiled with SDS sample buffer for 10 min. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore). The membranes were incubated in 5% powdered nonfat milk in TBST for 1 hour, and incubation with primary antibodies was done overnight at 4°C. Membranes were washed in TBST three times, followed by incubation with secondary antibodies (1:10,000; Abcam) for 1 hour at room temperature. After washing (TBST, 3x), immunoreactive products were detected by chemiluminescence and visualized and quantified with the ChemiDoc imaging system (Bio-Rad).
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