Cells were lysed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM DTT and complete proteinase inhibitors (Roche)) for 30 min at 4°C and centrifuged at 13000rpm for 15min. Protein concentrations were quantified by Bradford assay. We then added 4x sample buffer and boiled at 95°C for 10min. For immunoblotting, samples were loaded on 10% polyacrylamide gel and run until the appropriate protein separation was achieved. Samples were transferred onto the PVDF membrane, blocked for 1 hour in 5% non-fat milk in TBST (Tris-buffered saline + 0.1% Tween-20). The membranes were then incubated overnight at 4°C with the following primary antibodies: anti-Sox2 (R&D), anti-Nkx2-1 (Abcam) and Tubulin (Proteintech). After incubation with primary antibodies, the membranes were washed 3× in TBST then incubated with the appropriate HRP-labeled secondary antibody in TBST for 1 hour at room temperature. Membranes were then washed 3× with TBST then developed with regular ECL (Thermo Scientific). The intensity of individual bands was quantified using ImageJ.
Anti nkx2 1
Manufactured by Abcam
Sourced in United Kingdom
Anti-Nkx2-1 is a laboratory antibody product that recognizes the Nkx2-1 protein. Nkx2-1 is a transcription factor involved in the development and function of the thyroid, lung, and forebrain. This antibody can be used to detect and study the Nkx2-1 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by R&D Systems (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Anti phospho histone h2ax, by Cell Signaling Technology (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Anti cd31, by BD (1 mentions)
Anti p63, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Immunologic (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Pannoramic 250, by 3DHISTECH (1 mentions)
3 protocols using anti nkx2 1
1
Protein Extraction and Western Blot Analysis
2
Immunohistochemical Analysis of Lung Adenocarcinoma
3
Immunohistochemistry of Tissue Sections
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Immunohistochemistry was performed on paraffin-embedded sections (4 μm). Sections were dehydrated and antigenic epitopes were exposed by heating in 10-mM citrate buffer (pH 6.0) or by incubation in 0.05% trypsin at +37°C. Sections were incubated with the following antibodies: anti-NKX2-1 (Abcam, Cambridge, UK); anti-p63 (Abcam, Cambridge, UK); anti-EPHA3 (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA); anti-CD31 (Becton, Dickinson and Company, Franklin Lakes, NJ); anti-GFP (polyclonal rabbit serum 8 mg/ml, generated in house). Primary antibody staining was detected using Bright vision poly-horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ImmunoLogic, Duiven, The Netherlands), HRP-conjugated goat anti-rat IgG (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA) and 3,3′-diaminobenzidine (DAB) (Immunologic, Duiven, The Netherlands) or Alexa-Fluor-488-conjugated anti-rabbit IgG (Life Technologies/Thermo Fisher Scientific Inc., Waltham, MA). Sections were counterstained with Mayers hemalum solution (Millipore, Billerica, MA) or Hoechst 33342 dye (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA). Image acquisition was performed either using a Nikon 90i Eclipse microscope (Nikon Instruments Europe BV, The Netherlands) and DS-Fi2 5 MP camera, or a Pannoramic 250 3DHISTECH (3DHISTECH Kft., Budapest, Hungary) digital slide scanner with a 20× objective.
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