Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate

Samenvatting

Source: Rhinesmith, T. et al., Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples. J. Vis. Exp. (2017).

This video describes multimeric PAGE for separating native protein complexes from tissue homogenates. The technique is a hybrid of blue native-PAGE and SDS-PAGE techniques. The separated complexes can be characterized and studied for their role in cell functioning.

Protocol

1. Tissue Preparation

1. Prepare 10 mL of 4x BN-PAGE sample buffer (200 mM Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), 200 mM NaCl, 40% w/v glycerol, 0.004% Ponceau S, pH 7.2).
   NOTE: This stock solution may be made in advance and stored at 4 °C.
2. Dilute 250 μL of 4x sample buffer in 750 μL dH₂O containing 1x commercial protease inhibitor cocktail. Vortex and chill on ice.
3. Homogenize 20 mg of target tissue in the 1 mL 1x ice-cold BN-PAGE sample buffer with 30 strokes of a clean dounce homogenizer.
   NOTE: For this demonstration experi....

Resultaten

Figure 1: General multimer-PAGE flowchart.
(A) Prepare tissue lysate using non-denaturing methods, such as homogenizing in BN-PAGE sample buffer. (B) Pipette lysate into BN-P.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
<strong>Chemicals</strong>
&epsilon;-Aminocaproic acid&nbsp;	Sigma&nbsp;	A2504
Acrylamide&nbsp;	Acros Organics&nbsp;	164855000	Toxic.
Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)	BioRad&nbsp;	161-0148&nbsp;	Toxic.
Ammonium persulfate&nbsp;	Sigma&nbsp;	A3678
Anti rabbit IgG-HRP from goat	&nbsp;Santa Cruz Biotechnology&nbsp;	sc-2004
Bicinchoninic acid assay kit&nbsp;	Thermofisher&nbsp;	23225
Bis-tris&nbsp;	Sigma	&nbsp;B9754
Bovine serum albumin&nbsp;	Sigma	&nbsp;A9647
Butanol&nbsp;	Fisher Scientific&nbsp;	A399-1
Chemiluminescence substrate kit&nbsp;	ThermoFisher&nbsp;	24078
Coomassie blue G-250&nbsp;	Sigma-Aldrich&nbsp;	B0770
Digitonin	&nbsp;Sigma&nbsp;	D141&nbsp;	Toxic.
Dimethyl sulfoxide&nbsp;	Fisher Scientific&nbsp;	D128-1
Dithiobis(succinimidylpropionate)&nbsp;	Thermo Scientific	22585
Dry nonfat milk&nbsp;	LabScientific&nbsp;	M0841
Glycerol&nbsp;	Sigma&nbsp;	G9012
Glycine&nbsp;	Fisher Scientific&nbsp;	BP381-5
Halt Protease Inhibitor Cocktail&nbsp;	Thermofisher&nbsp;	78430
Hydrochloric acid&nbsp;	Fisher Scientific&nbsp;	A144SI-212&nbsp;	For titration. Caustic.
Methanol&nbsp;	Fisher Scientific&nbsp;	A412-4&nbsp;	For PVDF membrane activation. Toxic.
Monoclonal anti &alpha;-synuclein IgG from rabbit	Santa Cruz Biotechnology&nbsp;	sc-7011-R
N,N,N',N'-tetramethylethylenediamine	Sigma&nbsp;	T9281
N,N'-methylenebisacrylamide&nbsp;	Acros Organics&nbsp;	16479	Toxic.
NP40&nbsp;	Boston Bioproducts&nbsp;	P-872
Polysorbate 20 (tween-20)&nbsp;	Fisher Scientific&nbsp;	BP337-500
Polyvinylidene fluoride transfer membranes	Thermo Scientific&nbsp;	88518
Ponceau S&nbsp;	Sigma&nbsp;	P3504
Potassium chloride&nbsp;	Fisher Scientific&nbsp;	P217-3
Potassium phosphate monobasic	&nbsp;Sigma&nbsp;	P9791
Protease inhibitor cocktail&nbsp;	Thermo Scientific&nbsp;	88265
SDS solution (10% w/v)&nbsp;	BioRad&nbsp;	161-0416
Sodium chloride&nbsp;	Fisher Scientific&nbsp;	BP358-212
Sodium dodecyl sulfate&nbsp;	Sigma	&nbsp;L37771
Sodium phosphate monobasic&nbsp;	Fisher Scientific&nbsp;	BP329-1
Tricine&nbsp;	Sigma&nbsp;	T0377
Tris base&nbsp;	Fisher Scientific	&nbsp;BP152-500
Tris-HCl (0.5 M buffer, pH 6.8)&nbsp;	BioRad&nbsp;	161-0799
Tris-HCl (1.5 M buffer, pH 8.8)&nbsp;	BioRad&nbsp;	161-0798
<strong>Instruments</strong>
GE Imagequant LAS 4000	&nbsp;GE Healthcare&nbsp;	28-9558-10
ImageJ software NIH
Synergy H1 microplate reader&nbsp;	BioTek
Gel Former + Stand&nbsp;	Biorad
Microfuge 22R centrifuge&nbsp;	Beckman Coulter
2 mL dounce homogenizer
Vortex mixer&nbsp;	Fisher Scientific
Ultrasonic tissue homogenizer&nbsp;	Fisher Scientific&nbsp;	FB120220