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.
1. Microtubule-binding Assay
Table 1: Sample preparation for microtubule-binding assay.
Sample | Name | Microtubule needed | Main Protein needed | PEM-GTP-PTX |
1 | HCT 116-Control (6.21 μg/μl) | 2 μl | 9.66 μl (60 μg) | 108.34 μl |
2 | HCT 116-Curcumin 10 μM (4.81 μg/μl) | 2 μl | 16.63 μl (80 μg ) | 101.37 μl |
3 | HCT 116-Curcumin 20 μM (3.28 μg/μl) | 2 μl | 30.49 μl (100 μg) | 87.51 μl |
4 | HCT 116-LiCl 25 mM (5.43 μg/μl) | 2 μl | 18.42 μl (100 μg) | 99.58 μl |
5 | E. coli tau | 2 μl | 1 μl tau-352 | 117 μl |
6 | MT Only | 2 μl | X | 118 μl |
120 μl each |
The authors have nothing to disclose.
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 |