To evaluate the involvement of differential expression of telomerase protein (hTERT) in the gall bladder disease and carcinoma development, and its correlation with the mRNA expression status, the differential protein expression study was performed using western blotting method. Total protein was extracted using the Magnesium Lysis Buffer, and quantified using the BCA protein estimation kit. Proteins extracted from the disease (cholelithiasis and cholecystitis), carcinoma as well as non-neoplastic control cases (50μg each) were evaluated by western blotting analysis using antibody specific for telomerase (Y182 ab32020, Abcam). β-tubulin was used as loading control. Immunoflourescence study was done to evaluate the differential protein expression of gall bladder carcinoma and normal cases and validate the western blot analysis results, using antibody specific for telomerase (Y182 ab32020, Abcam) and goat anti-rabbit IgG HRP (Merck) secondary antibody.
Y182 ab32020
Manufactured by Abcam
Sourced in United Kingdom
Y182 ab32020 is a monoclonal antibody that targets the Y182 epitope. It is designed for use in various research applications, including immunohistochemistry and Western blotting. The core function of this product is to specifically bind to the Y182 epitope for detection and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
Axiovision, by Zeiss (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Goat anti rabbit igg hrp, by Merck Group (1 mentions)
4 protocols using y182 ab32020
1
Telomerase Expression in Gallbladder Disease
2
Western Blot Analysis of Telomerase Protein
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Cells were lysed in RIPA buffer (89901, ThermoFisher, Waltham, MA) supplemented with 1× protease inhibitor cocktail (PI78410, ThermoFisher, Waltham, MA) and then mixed with 4x LDS sample buffer (NP0008, ThermoFisher, Waltham, MA) and boiled at 95° C for 10 minutes. Samples were run on NuPAGE Novex 4–12% Bis-Tris Protein Gels (NP0335, ThermoFisher, ThermoFisher Scientific), and transferred to PVDF membranes. Membranes were incubated overnight at 4C with rabbit anti-human Tert antibody, (1:100 dilution) (Y182 ab32020, Abcam, Cambridge, MA) then HRP-linked anti-rabbit IgG antibody (1:2000 dilution) (7074S, Cell Signaling Technology, Danvers, MA) and visualized with Pierce ECL Western Blotting Substrate (32106, ThermoFisher, Waltham, MA) on the Bio-Rad ChemiDoc XRS+ System.
3
Subcellular Localization of Proteins
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For sub-cellular localization QT6 cells were transfected for 24 hours using Fugene HD transfection reagent (Promega, Charbonnières-les-Bains, France). After fixation, permeabilization and blocking steps, cells were incubated with a primary mouse anti-V5 antibody (R960-25; Invitrogen, Carlsbad, USA) or a rabbit anti-hTERT antibody (Y182, ab32020, Abcam, Cambridge, UK) for 1.5 hours followed by a goat anti-mouse antibody-Alexa Fluor 488 conjugate (A11029, Life Technologies, Saint-Aubin, France) or a goat anti-rabbit-Alexa Fluor 488 conjugate (A11008, Life Technologies, Saint-Aubin, France) for 45 minutes at room temperature. After washes, samples were mounted in DAPI-containing mounting medium (VECTASHIELD). Slides were analyzed by fluorescent microscopy (Axio observer Z1 and Axiovision, Carl Zeiss MicroImaging GmbH).
4
Immunohistochemical Analysis of TERT and DNA-PKcs
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The methods of have been described previously. 20 (link) In brief, resected liver specimens were xed in 10% v/v formalin, cut into blocks, and embedded in para n. The blocks were sliced into 4 µm-thick sections and stained with hematoxylin and eosin or used for immunohistochemical analysis. The TERT antibodies used for the analysis were a rabbit monoclonal antibody (Y182, ab32020, Abcam, UK) and a mouse monoclonal antibody (A-6, sc-393013, Santa Cruz, USA). The antibody for DNA-PKcs used was a mouse monoclonal antibody (#12311, Cell Signaling Technology, USA). The sections were subjected to dewaxing, heat-induced epitope retrieval with citrate buffer, antibody incubation (TERT: dilution 1:50, 15 minutes; DNA-PKcs: dilution 1:30, 45minutes) and counterstaining on a BOND Max immunostainer using Bond Epitope Retrieval Solution 2 (pH-9.0, 20 minutes) and the Bond Polymer Re ne Detection kit (Menarini, Berlin, Germany). The TERT expression was judged as positive when staining of the cytoplasm or the nucleus was observed in more than 30% of the tumor cells.
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