Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding 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 the microtubule-binding assay used to separate the microtubule-binding tau protein from non-binding hyperphosphorylated tau protein fractions in colorectal cancer cells. This assay helps determine the effect of various chemicals that can alter the phosphorylation status of tau protein, affecting its microtubule binding.
Protocol
1. Microtubule-binding Assay For the microtubule-binding assay, Add ~1 x 106 HCT 116 cells into 100 mm tissue-culture dishes containing the Minimum Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After one day of culturing, cells will reach 60-70% confluence. Prepare 1X (2 mL) PEM buffer from 10X PEM buffer stored at 4 °C by adding 20 µM (40 µL) paclitaxel and 1 mM (20 µL) GTP. Take the microtubule (…
開示
The authors have nothing to disclose.
Materials
| HCT 116 cell | ATCC | CCL-247 | |
| MEM (EBSS) | Hyclone | SH30024.01 | |
| 100 mm dish | Corning | 430161 | |
| Anti-Tau 13 antibody | abcam | ab19030 | |
| Dithiothreitol (DTT) | Roche | 10 708 984 001 | Storage Temperature 2–8 °C |
| Microlitre Centrifuges | Hettich Zentrifugen | MIKRO 200 R | |
| Paclitaxel | Sigma-Aldrich | T1912 | Storage Temperature 2–8 °C |
| Curcumin | Sigma-Aldrich (Fluka) | 78246 | Storage Temperature 2–8 °C |
| Microtubules (MT) | Cytoskeleton | MT001 | Store at 4 °C (desiccated) |
| Sodium hydroxide | Sigma | 72068 | |
| Magnesium Chloride | Sigma-Aldrich | M2670 | |
| GTP | Sigma-Aldrich | G8877 | Store at -20 °C |
| DPBS | WelGene | LB 001-02 | |
| Sonic Dismembrator | Fisher Scientific | Model 500 | |
| Ultracentrifuge | Beckman Coulter | Optima L-100 XP | |
| Bovine serum Albumin (BSA) | Sigma | A7906 | |
| Molecular Imager | Bio-Rad | ChemiDoc XRS+ | Store at 4 °C |
| Protein assay dye reagent | Bio-Rad | 500-0006 | |
| α-tubulin (11H10) Rabbit mAb | Cell signaling | 2125 | |
| GAPDH (14C10) Rabbit mAb | Cell signaling | 2118 | |
| Anti-Tau (phospho S396) antibody | Abcam | ab109390 | |
| Tau-352 human | Sigma | T 9950 | Store at -20 °C |
| PVDF membrane | Bio-Rad | 162-0177 | |
| Sodium Dodecyl Sulfate (SDS) | Affymetrix | 75819 | |
| Protein Assay | Bio-Rad | 500-0006 | Store at 4 °C |
| Goat anti-mouse IgG Secondary Antibody | ThermoFisher | A-11005 | Store at 4 °C in the dark |
