Total cellular protein was extracted from cell lines using RIPA lysis buffer (Beyotime Biotechnology). Protein concentration was detected using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime Biotechnology). The total protein was separated in a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel and then transferred to nitrocellulose membrane. After blocked with 5% non‐fat milk, the membrane was incubated with primary antibodies including anti‐GPX2 (#ab137431, Abcam, 1:1000), anti‐E‐cadherin (#3195, CST, 1:1000), anti‐vimentin (#5741, CST, 1:1000), anti‐Snail (#3879, CST, 1:1000), anti‐PI3K p85 (#4257, CST, 1:1000), anti‐phospho‐PI3K p85 (#4228, CST, 1:1000), anti‐AKT (#4691, CST, 1:1000), anti‐phospho‐AKT (#4060, CST, 1:1000), anti‐mTOR (#2983, CST, 1:1000), anti‐phospho‐mTOR (#5536, CST, 1:1000), and anti‐β‐actin antibodies (AC026, ABclonal, 1:10000). Second day, it was incubated with the Dylight 800‐conjugated secondary antibody (A23920, Abbkine, 1:500) or horseradish peroxidase‐conjugated secondary antibody, and the protein bands were visualized using Odyssey CLx Dual‐Color infrared Laser Imaging System (Li‐COR) or WesternBright ECL Kit (Advansta).
Ab137431
Manufactured by Abcam
Sourced in United Kingdom
Ab137431 is a lab equipment product. It is a tool used for laboratory research and analysis purposes. The core function of this product is to perform specific tasks within a laboratory setting. No further details on the intended use or interpretation of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot, by Bio-Rad (2 mentions)
Clarity ecl substrate, by Bio-Rad (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Westernbright ecl kit, by Advansta (1 mentions)
Ac026, by ABclonal (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Odyssey clx dual color infrared laser imaging system, by LI COR (1 mentions)
Image studio, by LI COR (1 mentions)
Ab34173, by Abcam (1 mentions)
Ab190685, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab124954, by Abcam (1 mentions)
Sc 166570, by Santa Cruz Biotechnology (1 mentions)
Beadblaster24r, by Benchmark Scientific (1 mentions)
Goat anti rabbit hrp, by Southern Biotech (1 mentions)
Mouse anti ecad, by BD (1 mentions)
Ab228649, by Abcam (1 mentions)
β actin antibody, by Merck Group (1 mentions)
5 protocols using ab137431
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Antioxidant Proteins
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Lung tissues were lysed in 1× RIPA buffer using a BeadBlaster24R (Benchmark Scientific, Sayreville, NJ, USA). Proteins were separated into 4–20% SDS-polyacrylamide gels and semi-dry-transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% milk and probed overnight on a 4 °C rocker with the following primary antibodies: anti-Gclc (ab190685, Abcam, Cambridge, UK), anti-Gpx2 (ab137431, 1:2000, Abcam), anti-Gpx4 (sc-166570, 1:1000, Santa Cruz, Santa Cruz, CA, USA), NQO1 (ab34173, 1:2000, Abcam), Sepp1 (PA5-112707, 1:1000, Invitrogen, Waltham, MA, USA) or Txnrd11 (ab124954, 1:1000, Abcam). Membranes were then incubated at room temperature for 1 h in secondary antibody of goat anti-mouse HRP (horseradish peroxidase) (1031-05, 1:2000, Southern Biotech, Birmingham, AL, USA) or goat anti-rabbit HRP (4030-05, 1:2000, Southern Biotech). Membranes were developed using enhanced chemiluminescence (ECL) with ChemiDoc imaging and quantified by densitometry using Image Studio (Li-Cor, v 5.2.1, Lincoln, Nebraska). All quantifications for western blots were normalized to β-actin (8H10D10, 1:2000 Cell Signaling Technology, Danvers, MA, USA).
3
Comprehensive Immunohistochemistry Antibody Panel
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Antibodies against GPx2 (AB137431), HIF1α (AB228649), NOTCH1 (AB52627), pAMPK (staining, AB23875) were obtained from Abcam. Rabbit polyclonal anti TWIST (sc-15393), was obtained from Santa Cruz Biotechnology. Rat anti KRT8 (staining, Cat# TROMA-I; RRID: AB_531826) was purchased from DSHB. Rabbit anti p63 (staining, 39692), VIM (staining and WB, 5741), SLUG (9585), E-CAD (staining and WB, 3195), pAMPK(WB, 2535), and mouse anti SNAI1 (3895) were from Cell Signaling and β-actin antibody was from Sigma. Rabbit anti KRT14 antibody (staining, 905304) was from Biolegend. Guinea pig anti KRT14 (staining, Cat#. GP-CK14) was from Progen. Mouse anti E-CAD (WB and staining, 610182) and mouse anti N-cad (staining, 610920) was from BD Transduction Labs. Rabbit anti-SLC2A1(GLUT1) antibody (WB, MBS9126610) was purchased from BioSource. Mouse anti-SLC2A1(GLUT1) (staining, NBP-75785-100ug) was purchased from Novus Biologicals. Alexa Fluor 405, 488, 594 goat anti mouse or rabbit or rat, Alexa Fluor 647 donkey anti guinea pig were from Invitrogen. Echinomycin was obtained from Tocris Bioscience.
4
Western Blot Analysis of Oxidative Stress Markers
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Samples were loaded onto 4–15% Criterion™ or Mini-PROTEAN® TGX™ gels (Bio-Rad), transferred to PVDF membranes (Trans-Blot®, Bio-Rad), blocked with 5% milk in Tris-buffered saline containing 0.05% Tween-20, and probed with anti-Nrf2 antiserum (1:1000; gift from Dr. Edward Schmidt, Montana State University), TXNRD1 antisera (1:2000, generated in collaboration with Dr. Edward Schmidt from full-length tag-less mouse TXNRD1 protein at Lampire Biological, Ottsville, PA), and GPX2 (1:2000, ab137431, Abcam) in 5% milk-TBST overnight. All antibodies were followed by goat anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology; 1:5000). Membranes were developed using Clarity™ ECL Substrate (Bio-Rad) and imaged using a ChemiDoc™ System (Bio-Rad). For loading control, membranes were reprobed with either anti-nucleolin antibody (ab22758, Abcam; 0.2 μg/mL) or anti-β-actin (sc-47778, Santa Cruz; 0.2 µg/mL).
5
Western Blot Analysis of GPX2 Expression
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Samples were loaded onto 4-20% Criterion or Mini-PROTEAN TGX gels (Bio-Rad), transferred to PVDF membranes (Trans-Blot, Bio-Rad), blocked with 5% milk in Tris-buffered saline containing 0.05% Tween 20 (TBST), and probed with rabbit anti-GPX2 (ab137431, Abcam; 1 : 1000 in 5% milk in TBST) followed by a goat anti-rabbit IgG-HRP secondary antibody (sc-2004; Santa Cruz Biotechnology; 1 : 5000 in TBST). Membranes were developed using Clarity ECL Substrate (Bio-Rad) and imaged using a ChemiDoc System (Bio-Rad). For loading control, membranes were reprobed with a mouse anti-β-actin antibody (sc-47778, Santa Cruz; 1 : 1000 in milk in TBST) followed by an anti-mouse IgG-HRP secondary antibody (HAF007; R&D Systems; 1 : 5000 in TBST). Pixel density of GPX2 was normalized to the respective actin band density.
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