MCF-7 cells were depleted of E2, as described above, preincubated overnight in estrogen-depleted medium at a density of 1 × 106 cells per dish, and then treated with QBMR (0.1 mg/mL) or 17β-estradiol (0.01 μM) with or without 0.1 μM ICI182, 780 and 0.1% DMSO treatment as negative control. All cells were incubated for 48 h and were harvested protein. The western blot protocol and semiquantitative analysis were carried out using the following protocol: ERα antibody (1:200, SC-542 Santa Cruz Biotechnology) and ERβ antibody (1:1000, ab3577, Abcam Biotechnology) were used, and GAPDH antibody (1:1000, SC-7907, Cell Signaling Technology) was used as internal control. All experiments were performed in triplicate. Mean normalized protein expression ± S.E. was calculated from independent experiments.
Ab3577
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab3577 is a primary antibody that can be used for the detection and quantification of a specific target protein in various laboratory applications. It is a polyclonal antibody produced in a suitable host animal. The core function of this product is to provide a tool for researchers to study the presence and levels of the target protein in their samples.
Automatically generated - may contain errors
Lab products found in correlation
Sc 542, by Santa Cruz Biotechnology (6 mentions)
Sc 7907, by Cell Signaling Technology (4 mentions)
Ab32063, by Abcam (4 mentions)
Ab181602, by Abcam (3 mentions)
Ab1376, by Abcam (2 mentions)
Ab2746, by Abcam (2 mentions)
Ab3523, by Abcam (2 mentions)
Sc 7907, by Santa Cruz Biotechnology (2 mentions)
Ab44520, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Tissue extraction reagent, by Thermo Fisher Scientific (1 mentions)
Densitometer, by GE Healthcare (1 mentions)
Fitc labeled goat anti rabbit igg h l a0562, by Beyotime (1 mentions)
Dp80 fluorescence microscope, by Olympus (1 mentions)
Ab92377, by Abcam (1 mentions)
Ab133526, by Abcam (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab39742, by Abcam (1 mentions)
Sc 7207, by Abcam (1 mentions)
19 protocols using ab3577
1
Evaluating Estrogenic Activity in MCF-7 Cells
2
Western Blot Analysis of Urethral Proteins
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Urethral protein samples were prepared by homogenization of cells in a tissue extraction reagent (Invitrogen, USA). Cell lysates containing 100 μg of protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred to a polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA, USA). The membrane was stained with Ponceau S to verify the integrity of the transferred proteins and to monitor the unbiased transfer of all protein samples. Detection of nNOS, iNOS, ERα, ERβ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the membranes was performed with an electrochemiluminescence kit (Amersham Life Sciences Inc., Arlington Heights, IL, USA) [28 (link)–30 (link)] using the following antibodies: goat polyclonal to nNOS, 1 : 300 dilution, Abcam (ab1376); rabbit polyclonal to iNOS, 1 : 200 dilution, Abcam (ab3523); mouse monoclonal to ERα, 1 : 1000 dilution, Abcam (ab2746); or rabbit polyclonal to ERβ, 1 : 500 dilution, Abcam (ab3577). The intensity of each band was quantified using a densitometer (Molecular Dynamics, Sunnyvale, CA).
3
Estrogen Receptor Expression in Uterus and Vagina
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The right horns of the uterus and vagina were stored at -80°C for western blot. Uterus and vagina were homogenized in RIPA lysis buffer supplemented with aprotinin on the ice for 30 minutes and then centrifuged at 15,000 rpm for 15 minutes at 4°C. Afterward, the supernatant was collected and protein concentration was estimated by Bradford. The dilution ratios of ERα (Abcam Biotechnology, ab32063), ERβ (Abcam Biotechnology, ab3577), and GAPDH (Abcam Biotechnology, ab181602) were 1:1000, 1:2000, and 1:50000, respectively. The blots were developed using an enhanced chemiluminescent assay. All experiments were done in triplicate. Alpha Ease FC (Fluorchem FC2) software was used to measure the relative quantity of each antibody [19 (link)].
4
Immunofluorescence Assay for ERβ
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U87-MG and C6 cells were seeded at a density of 2.5x105 cells/well on glass coverslips placed in 6-well tissue culture plates, and allowed to adhere overnight. Cells were then treated with 25 µM icaritin at 37˚C for 24 h. The following day, cells were washed with phosphate buffered saline (PBS) and fixed in 4% (v/v) paraformaldehyde for 30 min at room temperature. Fixed cells were subsequently washed with PBS and permeabilized with 0.3% (v/v) Triton X-100 in PBS for 1 h, prior to being washed again in PBS. In order to block non-specific binding sites, cells were incubated with PBS containing 5% (v/v) bovine serum albumin at room temperature for 10 min, and then incubated with a primary antibody against ERβ (1:5,000; diluted in 0.1% Triton X-100; ab3577; Abcam) overnight at 4˚C. The following day, cells were washed with PBS and incubated with a secondary antibody [1:500; diluted in mixture of 1% PBS containing 1% BSA and 0.3% Triton X-100; FITC-labeled goat anti-rabbit IgG (H+L); A0562; Beyotime Institute of Biotechnology] for 1 h in the dark. Cells were then gently washed. Finally, the cell nucleus was stained with DAPI (1 µg/ml; 4083; CST Biological Reagents Co., Ltd.) at room temperature for 10 min and visualized under a DP80 fluorescence microscope (x400 magnification; Olympus Corporation).
5
Quantifying Uterine and Vaginal Estrogen Receptors
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ERα and ERβ in uterus and vagina were examined by western blot, the protocol and semi-quantitative analysis were carried out following our previous study7 30 . The following antibodies were used: rabbit anti-estrogen receptor- α polyclonal antibody (1:200, SC-542, Santa Cruz Biotechnology), rabbit anti-estrogen receptor- β polyclonal antibody (1:1000, ab3577, Abcam Biotechnology), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:1000, SC-7907, Cell Signaling Technology, Danvers, MA, USA). The relative quantity of each antibody was measured by Alpha Ease FC (Fluorchem FC2) software. The density ratio of protein to GAPDH was calculated from the band density.
6
Immunohistochemical Analysis of ER-α, ER-β, PCNA, and Ki-67
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The immunohistochemistry protocol and semi-quantitative analysis were carried out as described in our previous study12 . The following antibodies were used12 23 (link)39 (link): rabbit anti-estrogen receptor-α polyclonal antibody (1:20, SC-542, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-estrogen receptor-β polyclonal antibody (1:50, ab3577, Abcam Biotechnology, Cambridge UK), rabbit anti-proliferating cell nuclear antigen (PCNA) polyclonal antibody (1:15, SC-7907, Santa Cruz Biotechnology), and rabbit anti-ki-67 polyclonal antibody (1:200, SC-7907, Santa Cruz Biotechnology) were used. The Image-Pro Plus 6.0 System image analysis system was used for quantitative analysis.
7
Evaluating QYFE's Estrogenic Effects
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MCF-7 cells were cultured in DMEM with 5% charcoal-dextran stripped FBS for 1 day to eliminate any estrogenic source before treatment, then the cells were treated with QYFE (0.00001~0.01mg/mL), 17β-estradiol (0.01 μM) with or without 0.1 μM ICI182, 780, 0.1% DMSO treatment as negative control. After 48h, all cells were harvested protein. Rabbit anti-ER α monoclonal antibody (dilution 1/1000, ab32063, Abcam Biotechnology, UK), rabbit anti-ERβ polyclonal antibody (dilution 1/2000, ab3577, Abcam Biotechnology, UK), rabbit anti-pS2 monoclonal antibody (dilution 1/1000, ab92377, Abcam Biotechnology, UK), rabbit anti-PR monoclonal antibody (dilution 1/1000, ab133526, Abcam Biotechnology, UK) were used, and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (dilution 1/5000, ab181602, Abcam Biotechnology, UK) was used as internalcontrol. Finally incubated with goat anti-Rabbit IgG polyclonal antibodyHRP-Labelled (1:4000; C1309; Beijing Pulilai Gene Technology Co., Ltd.). All determinations were carried out in triplicate. Visualization of protein bands was accomplished using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) [39 (link)].
8
Immunohistochemical Profiling of Lung
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Briefly, 5 µm sagittal sections of fixed human lung were deparifinized and rehydrated as discussed earlier. After epitope retrieval, oestrogen receptor (ER) alpha (ERα) (Santa Cruz, sc-7207; 1 µg/mL), ERβ (Abcam, ab-3577; 5 µg/mL), GPER (Abcam, ab-39742; 5 µg/mL), and SERT (Abcam, ab-44520: 1:200) were incubated overnight at 4°C. Lung sections were counterstained with haematoxylin. Distribution was assessed by staining consecutive sections with alpha smooth muscle actin (for medial cells) and von-Williebrand factor (for endothelial cells).
9
Quantification of Protein Expression by Western Blot
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Total protein was extracted using radio immunoprecipitation lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations were determined using a bicinchoninic acid assay kit (Beyotime Biotechnology). Proteins (30 μg) from each sample were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked in 5% defatted milk at room temperature for 1 h. Then, the membranes were incubated with mouse anti-human TET2 (1:400; ab94580, Abcam, Cambridge, UK), polyclonal rabbit anti-human ESR2 (1:400; ab3577, Abcam), polyclonal mouse anti-β-actin (1:30000; bs-2188R, Bioss, Beijing, China) antibodies at 4°C overnight and then with horseradish peroxidase–conjugated secondary anti-mouse/rabbit antibody at room temperature for 1 h. Proteins bands were visualized using an enhanced chemiluminescence system (Millipore) and were analyzed with ImageJ 2X (National Institutes of Health, Bethesda, MD). Protein levels were normalized to that of β-actin.
10
Muscle Protein Expression Analysis
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All muscle samples were tested using rabbit polyclonal anti-estrogen receptor alpha antibody, (orb13402) (Biorbyt®, United Kingdom), dilution 1:800; rabbit polyclonal anti-estrogen receptor beta, chIP grade (ab3577), (ABCAM®, United States), dilution 1:800; and rabbit polyclonal anti-androgen receptor antibody, chIP grade (ab74272), (ABCAM®, United States), dilution 1:200.
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