"
Team:TU Darmstadt/Protocols/SDS-Page
From 2012.igem.org
(Difference between revisions)
(Created page with "<html> <link rel="stylesheet" href="https://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/css&action=raw&ctype=text/css" type="text/css" /> <div id="TUD"> <div id="...") |
|||
| Line 27: | Line 27: | ||
<li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li> | <li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li> | ||
<li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li> | <li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li> | ||
| - | <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a | + | <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a></li> |
| - | + | ||
| - | + | ||
| - | + | ||
<li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul> | <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul> | ||
<li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li> | <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li> | ||
Latest revision as of 20:56, 26 September 2012
Contents |
SDS-Page
About
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge).[1]
SDS-PAGE (Laemmli)
Procedure
- prepare the separating gel and fill it into the chamber
- pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
- discard the isoprpyl alcohol and pour the prepared stacking gel
- stick in the comb
- store the gel in wet cloth (to prevent dehydration) at 4°C
Stacking Gel
For two gels
- 0.5 mL glycine
- 1.25 mL 0.5 M Tris buffer pH 6.8
- 0.025 mL 20% SDS
- 0.67 mL acrylamide
- 0.005 mL TEMED
- 0.025 mL APS
- 2.575 mL dist. water
Separating Gel
For two gels
- 3.0 mL glycine
- 7.5 mL 1.5 M Tris buffer pH 8.8
- 0.15 mL 20% SDS
- 12.0 mL acrylamide
- 0.02 mL TEMED
- 0.15 mL APS
- 7.2 mL dist. water
Buffer
Running buffer
- 3,03 g TRIS-HCl
- 15 g glycine
- 0,2 g SDS
- ad. 1 L dist. water
Staining buffer
- 7 mg coomassie brillant blue
- 200 mL acetic acid
- 1 L ethanol
- ad. 2 L dist. water
Sample buffer
- 4.8 g urea
- 0.242 g TRIS-HCl
- 0.2 g SDS
- 1 mg sodium bromphenolate
- ad. 10 mL dist. water
SDS-PAGE (Schägger)
Procedure
- same as above
Stacking Gel
For five gels
- 1.6 mL acrylamide
- 12.4 mL Tris-HCl/0,3% SDS pH 8.45
- 182 g TRIS-HCl
- 1,5 g SDS
- ad. 500 mL
- adjust pH 8.45
- 7.6 mL dist. water
- 100 µL 10% ammonium persulfate (APS)
- 10 µL TEMED
Separating Gel
For five gels
- 20.00 mL acrylamide
- 20.00 3 M Tris-HCl/0.3%SDS pH 8.45
- 182 g TRIS-HCl
- 1,5 g SDS
- ad. 500 mL dist. water
- adjust pH 8.45
- 100 µl 10% ammonium persulfate (APS)
- 10 µL TEMED
Buffer
Cathode buffer
- 12.11 g TRIS-HCl
- 17.92 g tricine
- 1 g SDS (0.1% SDS)
- ad. 1 L dist. water
Anode buffer
- 24.22 g TRIS-HCl
- ad. 1 L dist. water
- adjust to pH 8.9
Staining buffer
- 7 mg coomassie brillant blue
- 200 mL acetic acid
- 1 L ethanol
- ad. 2 L dist. water
Sample buffer
- 4.8 g urea
- 0.242 g TRIS-HCl
- 0.2 g SDS
- 1 mg sodium bromphenolate
- ad. 10 mL dist. water
Application
References
[1] http://en.wikipedia.org/wiki/SDS-page
