Whole cell lysates were prepared by resuspending a cell pellet in an appropriate amount of RIPA buffer containing protease inhibitors and phosphatase inhibitors and incubating for 30 minutes on ice. The lysates were spun down at high speed for 15 minutes at 4C, and the supernatant was transferred to a clean tube. Lysates were quantified using a Bradford assay (BioRad, 500-0205). 80 μg of total cell lysates were analyzed by Western blot using the following antibodies and concentrations: LRP6 (1:1000, Cell Signaling Technology 3395, RRID:AB1950408), LRP5 (1:1000, Cell Signaling Technology 5731, RRID:AB_10705602), ß-Catenin (1:1000, Cell Signaling Technology 8480, RRID:AB_11127855), ß-Tubulin (1:2000, Genetex GTX101279, RRID:AB_1952434). All blots were incubated overnight in primary antibody, washed, and then incubated for two hours in secondary antibody – anti-rabbit-HRP (1:5000, GE Healthcare) or anti-mouse-HRP (1:2000, GE Healthcare). Blots were imaged using a Syngene G-Box system. Bands were quantified using Adobe Photoshop (RRID:SCR_014199). Statistical evaluation of three or more independent biological replicates was performed using Student’s unpaired T-Test.
Gtx101279
Manufactured by GeneTex
Sourced in United States
GTX101279 is a laboratory equipment product offered by GeneTex. It is designed for sample preparation and analysis in biological research applications. The core function of this product is to facilitate the efficient extraction, separation, and purification of biological molecules, such as proteins, nucleic acids, or other biomolecules, from complex samples. Further details on the intended use or specific applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
G box system, by Syngene (1 mentions)
Anti mouse hrp, by GE Healthcare (1 mentions)
Catenin, by Cell Signaling Technology (1 mentions)
Anti rabbit hrp, by GE Healthcare (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Gtx113286, by GeneTex (1 mentions)
Sab2101014, by Merck Group (1 mentions)
Affinipure goat anti mouse fitc, by Jackson ImmunoResearch (1 mentions)
Affinipure goat anti rabbit rhodamine, by Jackson ImmunoResearch (1 mentions)
Vectashield antifade mounting medium h 1000, by Vector Laboratories (1 mentions)
Dmi 6000b cs, by Leica camera (1 mentions)
Enhanced chemiluminescence detection reagent, by Merck Group (1 mentions)
Gtx627408, by GeneTex (1 mentions)
3 protocols using gtx101279
1
Western Blot Analysis of Wnt Pathway Proteins
2
Immunofluorescent Staining of Cytoskeletal Proteins
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Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Samples were blocked for 30 min in PBS with 2% bovine serum albumin (BSA). Primary antibodies against β-tubulin (GTX101279, GeneTex, Irvine, CA, USA), γ-tubulin (GTX113286, GeneTex, Irvine, CA, USA), acetyl-α-tubulin (GTX16292, GeneTex, Irvine, CA, USA), and HAS3 (SAB2101014, Sigma-Aldrich, St. Louis, MO, USA) were diluted 1:100 in PBS with 1% BSA, and then incubated with the cells for 2 h at room temperature. AffiniPure goat anti-mouse FITC (15-095-003, Jackson ImmunoResearch, West Grove, PA, USA) and AffiniPure goat anti-rabbit rhodamine (111-025-144, Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies were diluted 1:50. Slides were incubated with secondary antibodies for 1 h at room temperature. Samples were mounted with VECTASHIELD Antifade Mounting Medium (H-1000, Vector Laboratories, Burlingame, CA, USA) and imaged via confocal microscopy (DMI 6000B CS, Leica, Wetzlar, Germany).
3
Western Blot Analysis of Apoptosis Markers
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The protein sample with × 5 loading buffer was boiled, electrophoresed on a 12% polyacrylamide gel, and transferred to PVDF membranes. Membranes were blocked with 5% skim milk or bovine serum albumin dissolved in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h, and incubated overnight at 4°C with rabbit anti-Bax (Cat No. 12789-1-AP, 1 : 5000, ProteinTech, US), rabbit anti-Bcl-2 (Cat no. 12789-1-AP, 1 : 2000, ProteinTech, US) or rabbit anti-cleaved caspase-3 (#9664, 1 : 1000, CST, US), anti-p-ERK1/2 (#4370, 1 : 1000, CST, US), anti-ERK1/2 (#4695, 1 : 1000, CST, US), anti-p-CREB (1 : 1000, #9198, CST, US), anti-CREB (#9197, 1 : 1000, CST, US) or anti-tubulin (GTX101279, 1 : 40000, GeneTex, US), and mouse anti-GAPDH (GTX627408, 1 : 10000, GeneTex, US) at 4°C, respectively. The next day, washed three times for 10 min in TBST, the membranes were incubated with appropriate secondary antibody for 1 h, and washed for another three times for 10 min at room temperature. Immunoreactive bands were observed with the enhanced chemiluminescence detection reagent (Millipore, USA) and analyzed using ImageJ software.
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