Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity

Abstract

Source: Hanna-Addams, S., et al. Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers. J. Vis. Exp. (2018).

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

1. 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.
   1. 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 3 min.
   2. After the cells are totally dissociated from the dish, add 10 mL of culture medium and transfer the cells with a 10-mL pipette to a 15-mL conical ....

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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.
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<br/> 10 mL glycerol<br/> 30 mL 5 M NaCl<br/> 840 mL ddH<sub>2</sub>O<br/> 10 mL Triton-X100<br/> (protease and phosphates inhibitors as desired)
10X TAE (1 L)			48.4 g Tris base<br/> 11.42 mL glacial acetic acid<br/> 20 mL 0.5M EDTA pH 8<br/> ddH20 to 1 L
4X SDD-AGE loading buffer (50 mL)			5 mL 10X TAE<br/> 10 mL glycerol<br/> 4 mL 20% SDS<br/> 0.5 mL 10% bromophenol blue<br/> 31 mL ddH<sub>2</sub>O
PBST Wash Buffer (1 L)			100 mL 10xPBS<br/> 800 mL ddH<sub>2</sub>O<br/> 1 mL Tween20
10X PBS (10 L)			800 g NaCl<br/> 20 g KCl<br/> 144 g Na<sub>2</sub>HPO<sub>4</sub>&middot;2H<sub>2</sub>O<br/> 24 g KH<sub>2</sub>PO<sub>4</sub><br/> add ddH<sub>2</sub>O to 10 L