This video demonstrates the two-dimensional semi-denaturing detergent agarose gel electrophoresis-based separation of amyloid fibers. The technique separates the polymorphic amyloid aggregates based on their heterogeneous size.
Protocol
1. Prepare Samples Culture 2 x 106 amyloid producing HT-29 colon cancer cells in a 10-cm tissue culture dish in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and penicillin-streptomycin. Culture cells overnight in a 37 °C incubator with 5% CO2. After the cells grow to 80% confluency, wash the cells with 10 mL of phosphate-buffered saline (PBS). Add 3 mL of Trypsin solution and incubate at 37 °C for …
Representative Results
Figure 1. Experimental protocol. (A) Schematic of the two-dimensional semi-denaturing detergent agarose gel electrophoresis (2D SDD-AGE). Begin by loading the sample in the right most lane, labeled in red. After termination of the first dimension run, rotate the gel 90° counter-clockwise. Run the second dimension electrophoresis. Solid arrow indicates the direction of the first dimension …
Divulgazioni
The authors have nothing to disclose.
Materials
gel electrophoresis unit
Fisher
HE99XPRO
appratus for gel running.
agrose
VWR
97062-250
For agarose gel.
DMEM
Sigma
D6429
for cell culture
fetal bovine serum
Sigma
F4135
for cell culture
penicilin-streptomycin
Sigma
P4333
for cell culture
Trypsin solution
Sigma
T4049
for cell culture
PBS for tissue culture
Sigma
D8662
for cell culture
recombinant TNF
made in our lab
for inducing necroptosis. See reference 11.
smac-mimetic
gift from Dr. Xiaodong Wang
for inducing necroptosis. See reference 11.
ZVAD-FMK
ApexBio
A1902
for inducing necroptosis. See reference 11.
Cell Counter
Bio-Rad
1450102
Model TC20; for counting cells
Pierce™ BCA Protein Assay Kit
Thermo Scientific
23225
for measuring protein concentration in cell lysates
Cell lifter
Fisher
07-200-364
to remove cells from dish
Lysis Buffer (1 L)
20 mL 1 M Tris pH 7.4
10 mL glycerol
30 mL 5 M NaCl
840 mL ddH2O
10 mL Triton-X100
(protease and phosphates inhibitors as desired)
10X TAE (1 L)
48.4 g Tris base
11.42 mL glacial acetic acid
20 mL 0.5M EDTA pH 8
ddH20 to 1 L
4X SDD-AGE loading buffer (50 mL)
5 mL 10X TAE
10 mL glycerol
4 mL 20% SDS
0.5 mL 10% bromophenol blue
31 mL ddH2O
PBST Wash Buffer (1 L)
100 mL 10xPBS
800 mL ddH2O
1 mL Tween20
10X PBS (10 L)
800 g NaCl
20 g KCl
144 g Na2HPO4·2H2O
24 g KH2PO4
add ddH2O to 10 L
Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity. J. Vis. Exp. (Pending Publication), e21109, doi: (2023).