SDS_PAGE-原理和方法

2.
The Principle
About DTT/ME
1,4-dithiothreitol (DTT) or 2-mercaptoethanol (ME)are used to cleave disulfide bonds of proteins.
DTT/Me
SH
S-S HS
The Principle
Migration of band (cm)
Relative migration/mobility
Rf =
Migration of dye front (cm)
The Procedure
1. Preparation of the gel Reagents
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
A Quick Intro to the SDS-PAGE
1. 2. Stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins are separated using polyacrylamide gels rather than agarose gels, because the polyacrylamide matrix: A) have pore sizes similar to the sizes of proteins (DNA is usually larger in size) B) are much tighter than agarose gels, thus are able to resolve the smaller protein molecules. 3. 4. Separates proteins according to molecular weight. Prior to electrophoresis, proteins are treated with sample loading buffer containning sodium dodecyl sulfate and dithiothreitol.
The Principle
About SDS
1.
linearlizes the protein structure, eliminates the effect of differences in shape . gives the protein an overall negative charge with a strength that is relative to the length of the protein. SDS coats proteins with approx. uniform charge-to-mass ratio (approx. 1.4g SDS/g protein).
Proteins Separation by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
He Zhiliang Chongqing Medical University November 13, 2010
Workshop Outline
A. B. C. The Apparatus used in the experiment. An Intro to the SDS-PAGE The principle: C1. About the gel C2. About SDS C3. About DTT C4. MW estimation and standard curve The Procedure: D1. preparation of the gel D2. sample preparation D3. running the gel D4. protein detection by stainning Notes Discussion
Proteins treated with sample loading buffer containning SDS and Me
The PriLeabharlann Baiduciple
MW estimation: standard curve Proteins separation by SDS-PAGE is due solely to differences in size, so, this method can be used to determine MW of proteins. A calibration curve can be made by plotting relative mobility of standard proteins ( i.e. mobility relative to bromophenol blue track dye)versus their log MW, and subsequently used to estimate the MW of an unknown protein running on the same gel.
2
A lower separating (ruuning) gel with higher concentration(7%~15% ) of gel and high pH(8.8) → separates according to molecular weight.
http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg
The Procedure
1) Clean and completely dry the plates, combs, and any other experiment materials. Assemble the system according to manufacturer’s instruction. 2) Prepare the separating gel solution as directed in the tabe of last page. Ammonium persulfate and TEMED are added when the gel is ready to be polymerized, stir gently, then immediately apply to the sandwich until the gel height is lower 1.5 cm than the short plate. Allowing the gel to polymerize 30~60min by covering gently with a layer of water (1cm). 3) Until a sharp optical discontinuity at the watet/gel interface is visible, pour off the water. 4) Prepare the stacking gel solution in a similar way to the separating gel . Insert the comb and allow the gel to polymerize completely.
The Procedure
Recipes for seperating and stacking gels
Reagents Distilled water 1.5 mol/L Tris-Cl buffer, pH 8.8 0.5 mol/L Tris-Cl buffer, pH 6.8 30% Acrylamide stock solution 10% (W/V) SDS 10% (W/V) APS 10% (W/V) TEMED Total 6.0 0.15 0.15 0.1 15 12% Seperation Gel (ml) 4.8 3.8 1.25 0.83 0.05 0.05 0.1 5 5% Stacking Gel (ml) 2.72
Chloride ions move faster (leading ions) than glycine (trailing ions) and create steep voltage gradient. Glycine ions are pulled due to the charge gradient and the two ions move at same speed. The proteins with intermediate mobility are carried along becoming stacked into very thin, distinctive layers in order of mobility.
The Principle
polymerization
The Principle
A discontinuous gel system is used frequently
1. An upper stacking gel with low concentration (2.5%~5% ) and low pH(6.8) →
The Principle
About the gel Polyacrylamide gels (PAG)are formed by the
polymerization of acrylamide in aqueous solution in the presence of small amounts of a bifunctional crosslinker, usually bisacrylamide (bis). The polymerization occurs via a free-radical mechanism after adding a catalyst. 1) ammonium persulfate initiates free radicals 2) tertiary aliphatic amine N,N,N',N'-tetramethylethylenediamine (TEMED) is a catalyst for free radical propagation The copolymerization a mesh-like network in three dimensions, consisting of acrylamide chains with interconnections formed from the bisacrylamide. Gel pore size is determined by the total acrylamide concentration and degree of cross-linking that regulated by varying ratios of acrylamide to bisacrylamide.
MW estimation: standard curve
standard unkown proreins prorein
Dye front Band
LgMW(KD) Unknown protein
+
(Rf)
dye front
Relative mobility Standard curve for protein MWs by SDS-PAGE
30% Acrylamide stock solution. 1.5 mol/L Tris-Cl buffer, pH 8.8. 0.5 mol/L Tris-Cl buffer, pH 6.8. 10% (W/V) SDS. 10% (W/V) Ammonium persulfate (APS). 10% (W/V) TEMED. distilled water. 2× sample loading buffer. × 5×ADS-PAGE running buffer, pH 8.8. × solutions for Commassie staining and destaing. protein marker.
D.
E. F.
The Apparatus
http://www.gbiosciences.com/Educational Uploads/EducationalProductsImages/mediumimages/Protei n%20Electrophoresis.jpg
http://www.nda-analytics.com/images/pipetting.jpg