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

  1. 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.
  2. 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 (MT) stock from -80 °C freezer and the E. coli tau working stock from -20 °C freezer.
  3. Prepare samples as per Table 1.
  4. Incubate at 37 °C for 30 min. Ultracentrifuge at 25 °C for 60 min at 100,000 x g.
  5. Transfer 120 µL of supernatants containing unbound tau, to clean and labeled tubes.
  6. Add 120 µL (same volume as supernatant) of 5X sample buffer to the pellet containing bound tau and mix thoroughly. Transfer the solution to clean labeled tubes and mix thoroughly.
  7. Measure the protein concentration by the Bradford assay using commercial protein-assay kits.
    NOTE: While preparing samples, use the volume of solution (step 1.6) for preparing both the pellet and supernatant samples.
  8. Prepare samples of equivalent protein concentration and add freshly prepared 4x SDS gel-loading buffer containing DTT (400 mM).
    NOTE: Samples can be stored at -20 °C at this stage.
  9. Incubate the samples on a heat block at 100 °C for 5 min. Mix the samples using a vortex. Cool at room temperature for ~10-15 min.
  10. Assemble an electrophoresis system for running a 10% polyacrylamide gel by SDS-PAGE.
    1. Load 10 µg protein (13 µL sample) per well on a 10% polyacrylamide gel. Load the molecular-weight ladder.
    2. After loading the sample, connect the positive and negative electrodes of the electrophoresis system to a power source to maintain a constant voltage. Initially, start at 70 V for 20 min to move the protein samples from the stacking gel into the resolving gel. Then, increase the voltage to 125 V and run the gel for approximately 70-120 min until samples and protein ladder reach the end of the gel.
    3. After completion of electrophoresis, transfer the gel onto a PVDF membrane using a wet electroblotting system. Transfer for ~100 min at 100 V.
    4. Mark the blot by making a small cut on the side of the molecular-weight ladder. Use a transparent plastic sheet and a sharp cutter for cutting the blot.
  11. Block the membrane in 4% BSA blocking buffer for 90 min at room temperature.
  12. Incubate the blot in primary-antibody solution (anti-tau at 1:5,000) at 4 °C overnight. Wash the blot in PBST four times, for 7 min each.
  13. Prepare the relevant secondary-antibody solution (goat anti-mouse IgG at 1:5,000), and incubate the blot for 90 min at room temperature. Wash in PBST four times, for 7 min each.
  14. Develop the blot using a chemiluminescence kit according to the manufacturer's recommendations. Cover the blot with transparent plastic wrap. Acquire images by a chemiluminescence imaging system for chemiluminescence with relevant software.

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

Divulgazioni

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

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Citazione di questo articolo
Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding Tau Protein in Colorectal Cancer Cells. J. Vis. Exp. (Pending Publication), e20340, doi: (2023).

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