The slides were baked at 60 °C for overnight, dewaxed in xylene and rehydrated with gradient alcohol, then the slides were boiled in citrate buffer for antigen retrieval. After that, the slides were immersed in 3 % H2O2 to inactivate endogenous peroxidase and blocked with goat serum for 30 min. Next, the slides were incubated with the primary monoclonal against MTA1 (ab71153; Abcam, Cambridge, MA, USA) and EpCAM (A1177; ABclonal, Boston, USA) diluted in 1:400 and 1:200 respectively in PBS at 4 °C, followed by incubation with biotinylated-second antibodies and streptavidin-peroxidase complex. And protein staining signal was then observed with 3′-diaminobenzidine. Lastly, the slides were counterstained with hematoxylin and mounted with neutral balsam. For a negative control, the slides were incubated with PBS instead of the primary antibody.
Ab71153
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab71153 is a monoclonal antibody targeting the protein PCNA. It is suitable for use in various immunoassay applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (4 mentions)
Ab15148, by Abcam (3 mentions)
Mab8969, by R&D Systems (2 mentions)
Ab180630, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
Ba1034, by Thermo Fisher Scientific (1 mentions)
Ba1074, by Thermo Fisher Scientific (1 mentions)
Ab207303, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Enhanced ripa lysis buffer, by Leagene (1 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
Ab73734, by Abcam (1 mentions)
Ab227981, by Abcam (1 mentions)
Loading buffer, by Bio-Rad (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
22 protocols using ab71153
1
Immunohistochemical Analysis of MTA1 and EpCAM
2
Western Blot Analysis of Protein Expression
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The western blot assays were performed as described in a previous article [23 (link)]. Total proteins of cells and tissues were collected and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then bound to PVDF membrane (Reno, Hangzhou, China). Following this step, 5% non-fat milk was used to seal the membranes for 1.5 hours, and the membranes were hybridized to anti-α1-AT (Abcam, ab207303, 1: 1200), anti-E-cadherin (Abcam, ab15148, 1: 1000), anti-TIMP-2 (Abcam, ab180630, 1: 1000), anti-MTA1 (Abcam, ab71153, 1: 800), anti-MMP2 (Abcam, ab92536, 1: 1500), anti-phosphorylated-mammalian target of rapamycin (p-mTOR) (Invitrogen, 710216, 1;800), anti-mTOR (Invitrogen, 44-1125G, 1: 1000), anti-phosphorylated-protein kinase B (p-Akt) (Invitrogen, 44-623G, 1: 1200), anti-Akt (Invitrogen, 44-623G, 1: 1600), anti-phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) (Invitrogen, PA5-12799, 1: 1600), anti-PI3K (Invitrogen, MA5-17149, 1: 1000), anti-β-actin (R&D, MAB8969, 1: 2000). After hybridization, the membrane was soaked in corresponding secondary antibodies (HRP mouse ant-goat IgG, Invitrogen, BA1074, 1: 7000; HRP mouse anti-rabbit, Invitrogen, BA1034, 1: 7000) at 37°C for 60 minutes. The protein was detected by ECL detection reagent (Taixin, Beijing, China).
3
Protein Profiling of Cell Signaling
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Total proteins of the tissues and cells were harvested and lysed with an enhanced RIPA lysis buffer (Leagene, Beijing, China). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%SDS-PAGE). Then, the protein was bound to the PVDF membrane (Hongda; Zhuzhou, Guangdong China). Subsequently, 5% skim milk was used to seal the membranes for 1.5 h, and membranes then were incubated with anti-STOML2 (Abcam, EP18708, 1: 1000), anti-E-cadherin (Abcam, ab15148, 1: 1200), anti-tissue inhibitor of metalloproteinases2 (TIMP2) (Abcam, ab180630, 1: 800), anti-metastatic tumor antigen 1 (MTA1) (Abcam, ab71153, 1: 1000), anti-matrix metalloproteinase-2 (MMP-2) (Invitrogen, MA1-772, 1: 1200), anti-MMP-9 (Invitrogen, PA5-13199, 1: 1000), anti-NF-κB (Abcam, E379, 1: 800), anti-IκB (CST, 9242S, 1: 1000), and anti-β-actin (R&D, MAB8969, 1: 2000) at 4°C in a refrigerator for 24 h. On the next day, the membranes were incubated with the secondary antibodies (Goat anti-mouse IgG, Abcam, ab7064, 1: 8000; Rabbit anti-mouse IgG, CST, #58802, 1: 7000; Mouse anti-rabbit IgG, CST, #3678, 1: 8000) at 37°C for 60 min. Proteins were analyzed by use of the iBright™ imaging system (A32749, Thermo, Shanghai, China).
4
Western Blot Analysis of Protein Targets
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Total proteins were boiled for 5 min and mixed with loading buffer (Bio-Rad, Shanghai, China) and then loaded onto SDS-polyacrylamide gel for electrophoresis. The SDS-PAGE was put into the electrophoresis apparatus with 100 V for 2 h and then transferred to a PVDF membrane. The membrane was blocked in 5% non-fat milk with PBST (Solarbio Life Sciences, Beijing, China) at room temperature for 2 h. Anti-LYRIC (MTDH) antibody (ab227981, Abcam, San Francisco, USA, 1: 2000, 64 kDa), Anti-E-Cadherin antibody (ab15148, Abcam, San Francisco, USA, 1: 2000, 120 kDa), Anti-MTA1 antibody (ab71153, Abcam, San Francisco, USA, 1: 2000, 81 kDa), anti-matrix metalloproteinase (MMP) −2 antibody (ab37150, Abcam, San Francisco, USA, 1: 2000, 72 kDa), anti-MMP-9 antibody (ab73734, Abcam, San Francisco, USA, 1: 2000, 95 kDa), and anti-GAPDH antibody (ab181602, Abcam, San Francisco, USA, 1: 2000, 36 kDa) were used separately to detect the target proteins with shaking overnight. The membranes were then washed with PBST 3 times with shaking. The membrane was probed with the secondary antibody IgG H&L (HRP) (ab6721, Abcam, San Francisco, USA, 1: 2000) for 1 h at room temperature. Pierce™ ECL plus Western blotting substrate (Thermo Fisher, Waltham, USA) was used to detect the blots and images were taken for analysis.
5
Immunohistochemical Analysis of MTA1 and Ki-67
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Tumor tissues were fixed with 4% paraformaldehyde (Beijing solarbio biotechnology co., LTD.). The sections (4 μm) were dewaxed as usual. After inactivating endogenous peroxidase, the sections were heated to repair antigen. The primary MTA1 antibody (1:200, ab71153, Abcam) and Ki-67 antibody (1:200, ab15580, Abcam) were cultured at 4 °C for overnight. After washing with PBS, biotin-labeled MTA1 secondary antibody (1:800, 85–9043, HRP, Broad Spectrum) was added for 1 h. DAB was used for coloring and Beyotime Biotechnology was used for re-dyeing. Results were observed with an optical microscope.
6
Protein Expression Analysis in Cell Lysates
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Total proteins were extracted via RNA immunoprecipitation (RIPA) buffer (Beyotime). Protein samples were isolated using 10%–15% SDS‐PAGE and then transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime). Subsequently, the PVDF membrane was sealed using 5% skim milk and cocultured with primary antibodies including anti‐Bcl‐2 (1:1000, ab59348, Abcam), anti‐Bax (1:1000, ab182733, Abcam), anti‐GLUT1 (1:1000, ab150299, Abcam), anti‐HK2 (1:1000, ab209847, Abcam), anti‐MTA1 (1:1000, ab71153, Abcam) and anti‐β‐actin (1:1000, ab6276, Abcam). The membrane was then coincubated with secondary antibody (IgG H&L, 1:20000, ab205718, Abcam). The protein signal was visualized using enhanced chemiluminescence (ECL) reagent (Beyotime).
7
Western Blot Analysis of MTA1 Protein
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The expression of MTA1 protein in each group was detected by western-blot. The primary antibodies were Anti-MTA1 Antibody (1:2000, ab71153, Abcam, UK) and beta-actin polyclonal Antibody (1:1000, ab8227, Abcam, UK). The second antibody was goat anti-rabbit Ig G (1:2000, ab6721, Abcam, UK) which needed to incubate for 1 h. After washing, protein bands were made color by ECL for 3-5 min. Image J (NIH) software was used for gray scale scanning and quantification.
8
Protein Expression Analysis via Western Blot
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Cells were lysed in RIPA buffer, and the protein concentration was measured using the Bradford assay. The whole protein samples were loaded onto 10 % SDS-PAGE gels, and were transferred to a PVDF membrane after electrophoresis. The membrane was blocked with 5 % fat-free milk, and then incubated with anti-MTA1 polyclonal antibody (ab71153; Abcam, Cambridge, MA, USA), anti-EpCAM polyclonal antibody (A1177; ABclonal, Boston, MA, USA) and anti-β-actin monoclonal antibody (A5316; Sigma-aldrich, St. Louis, MO, USA) overnight at 4 °C, followed by peroxidase-conjugated secondary antibody (1:5000) incubation for 1 h at room temperature. Signals were visualized with ECL and detected using LAS 4000 Imaging system (Fijifilm, Tokyo, Japan).
9
Western Blot Analysis of MTA1, HDAC1, and Nur77
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MH7A cells or synovial tissues were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The total protein concentration was quantified by using BCA Protein Kit (Beyotime, Shanghai, China). Then, equal amount of protein was separated on 10% SDS-PAGE, and the protein bands were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA).The membranes were blocked with 5% nonfat milk for 2 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-MTA1 antibody (1 : 1000, ab71153, Abcam), rabbit polyclonal anti-HDAC1 antibody (1 : 1000, ab19845, Abcam), rabbit polyclonal anti-Nur77 antibody (1 : 1000, ab13851, Abcam), and rabbit polyclonal anti-GAPDH antibody (1 : 2500, ab9485, Abcam). Then, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (1 : 2000, Abcam, ab6721) for 1 h. GAPDH was used as an endogenous control. The protein bands were visualized with ECL detection reagents and analyzed with the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
10
Immunofluorescence Staining of MTA1 in Cells
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For immunofluorescence staining, cells were seeded into 24-well plates with glass coverslips and fixed with 4% paraformaldehyde in PBS. Cell membranes were permeabilized with 0.5% Triton X-100 in PBS, and nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) in PBS. Slides were incubated with the first antibody, MTA1 (Abcam, ab71153; 1:200), at 4°C overnight. After three washes with cold PBS, cells were incubated with Cy3-conjugated IgG (EK022, 1:200 dilution in PBS) at room temperature for 30 min. After three washes with cold PBS, cells were sealed with a fluorescence quenching sealing tablet containing DAPI (36308ES11, Yeasen) and examined under a confocal microscope (Olympus, Tokyo, Japan)., TRITC Phalloidin (100 nM; 40734ES75, Yeasen, Shanghai, China) was used for cell structure staining.
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