Phosphatase Assay in CRC Cells: A Method to Examine the Phosphorylation Status of Tau Protein in Colorectal Cancer Cells
Published: April 30, 2023
Abstract
Source: Huda, M. N., et al. Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells. J. Vis. Exp. (2017)
This video describes a protocol for assessing the phosphorylation status of tau protein by a phosphatase assay in colorectal cancer cells. Phosphatase enzyme acts on the phosphate groups present on the tau protein, increasing the mobility of tau protein on SDS PAGE gel compared to the untreated tau protein samples.
Protocol
1. Phosphatase Assay Treat the cell lysates when cells reach ~65% confluence (approximated using microscopy), remove the MEM supplemented medium and add serum-free MEM with 5 µM, 10 µM, and 30 µM curcumin or 25 mM, 50 mM, and 100 mM LiCl. Incubate at 37 °C for 24 h in a humidified atmosphere containing 5% CO2, 24 h after treatment, wash the cells with 1 mL ice-cold phosphate-buffered saline (PBS), and scrape the cells using a cell scraper. Transfer the scraped c…
Açıklamalar
The authors have nothing to disclose.
Materials
| HCT 116 cell | ATCC | CCL-247 | |
| 100 mm dish | Corning | 430161 | |
| MEM (EBSS) | Hyclone | SH30024.01 | |
| Fetal Bovine Serum (FBS) | ThermoFisher (Gibco) | 16000044 | Store at -20 °C |
| Anti-Tau 13 antibody | Abcam | ab19030 | |
| Dithiothreitol (DTT) | Roche | 10 708 984 001 | Storage Temperature 2–8 °C |
| Curcumin | Sigma-Aldrich (Fluka) | 78246 | Storage Temperature 2–8 °C |
| Anti-Tau (phospho S396) antibody | abcam | ab109390 | |
| FastAP Thermosensitive Alkaline Phosphatase | Thermo Scientific | EF0651 | Store at -20 °C |
| PMSF | Sigma | P7626 | Store at room temperature |
| Phosphatase Inhibitor Cocktail | Cell Signalling | 5870 | Store at 4 °C |
| Protease Inhibitor Cocktail | Cell Signalling | 5871 | Store at 4 °C |
| Goat anti-mouse IgG Secondary Antibody | ThermoFisher | A-11005 | Store at 4 °C in the dark |
| Sodium Dodecyl Sulfate (SDS) | Affymetrix | 75819 | |
| Molecular Imager | Bio-Rad | ChemiDoc XRS+ | Store at 4 °C |
| RIPA Buffer | Sigma | R 0278 | Storage Temperature 2–8 °C |
| Triton X-100 | Sigma-Aldrich | X – 100 | Store at around 25 °C |
| PVDF membrane | Bio-Rad | 162-0177 | |
| Goat anti-mouse IgG Secondary Antibody | ThermoFisher | A-11005 | Store at 4 °C in the dark |
| Sodium Dodecyl Sulfate (SDS) | Affymetrix | 75819 | |
| Protein assay dye reagent | Bio-Rad | 500-0006 | |
| Sonic Dismembrator | Fisher Scientific | Model 500 | |
| Microlitre Centrifuges | Hettich Zentrifugen | MIKRO 200 R |
Bu Makaleden Alıntı Yapın
Phosphatase Assay in CRC Cells: A Method to Examine the Phosphorylation Status of Tau Protein in Colorectal Cancer Cells. J. Vis. Exp. (Pending Publication), e20339, doi: (2023).