SDS sample buffer

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==Protein gel sample loading buffer==
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==Purpose==
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===Purpose===
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For preparation and loading of protein samples onto a gel for SDS-PAGE analysis (Western blot/protein blot).
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*For preparation and loading of protein samples onto a gel for SDS-PAGE analysis.
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===Preparation===
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* Preparation: SDS contained in the sample buffer makes proteins negatively charged proportionally to their length. 2-mercapto-ethanol/DTT breaks disulphide bonds.
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To make 10 mL of 4X stock:
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* Loading: glycerol makes the sample buffer more dense than the surrounding running buffer of the protein gel, enabling easy loading into the gel pockets.
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==Preparation==
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====To make 10 mL of 4x stock====
* 2.0 ml 1M Tris-HCl pH 6.8  
* 2.0 ml 1M Tris-HCl pH 6.8  
*0.8 g [[SDS]]  
*0.8 g [[SDS]]  
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*8 mg bromophenol Blue  
*8 mg bromophenol Blue  
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Final concentrations (1X):
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====Final concentrations (1x)====
*50 mM Tris-HCl pH 6.8  
*50 mM Tris-HCl pH 6.8  
*2% [[SDS]]
*2% [[SDS]]
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*0.02 % bromophenol blue
*0.02 % bromophenol blue
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===Use===
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Dilute protein sample 1:3 into 4X sample loading buffer. Load on acrylimide gel in SDS-PAGE buffer.
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==Use of loading buffer==
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===Safety===
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Dilute protein sample 1:3 into 4X sample loading buffer. Load on acrylimide gel in SDS-PAGE buffer.
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==Safety==
β-mercaptoethanol is a severe irritant and is readily absorbed through the skin.  [[SDS]] is a respiratory irritant in solid form and a mask should be worn while weighing it.
β-mercaptoethanol is a severe irritant and is readily absorbed through the skin.  [[SDS]] is a respiratory irritant in solid form and a mask should be worn while weighing it.
*[http://ptcl.chem.ox.ac.uk/MSDS/ME/2-mercaptoethanol.html β-mercaptoethanol MSDS]
*[http://ptcl.chem.ox.ac.uk/MSDS/ME/2-mercaptoethanol.html β-mercaptoethanol MSDS]
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===References===
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==References==
*[http://genetics.mgh.harvard.edu/szostakweb/resources/Public%20Protocols/protein_page/index.html Szostak Lab SDS-PAGE]
*[http://genetics.mgh.harvard.edu/szostakweb/resources/Public%20Protocols/protein_page/index.html Szostak Lab SDS-PAGE]
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==Links==
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* 5x SDS loading buffer [http://mach7.bluehill.com/proteinc/tutorial/sdspage.html]
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* SDS loading buffer, Lynn lab, Uni Kentucky [http://www.google.de/url?sa=t&rct=j&q=sds%20page%20loading%20buffer&source=web&cd=15&ved=0CEEQFjAEOAo&url=http%3A%2F%2Fwww.chem.uky.edu%2Fresearch%2Flynn%2FSept%252018.pdf&ei=9GCpTvixAsWDhQeKpoT9DQ&usg=AFQjCNFF620xaiEhOJerQPKwnEVFjY7Apg&cad=rja]
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* Wikipedia: [http://en.wikipedia.org/wiki/SDS-PAGE SDS-PAGE], [http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate SDS]
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[[Category:Material]]
[[Category:Material]]
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[[Category:Buffer]]
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[[Category:Buffers]]

Current revision

Contents

Purpose

For preparation and loading of protein samples onto a gel for SDS-PAGE analysis (Western blot/protein blot).

  • Preparation: SDS contained in the sample buffer makes proteins negatively charged proportionally to their length. 2-mercapto-ethanol/DTT breaks disulphide bonds.
  • Loading: glycerol makes the sample buffer more dense than the surrounding running buffer of the protein gel, enabling easy loading into the gel pockets.


Preparation

To make 10 mL of 4x stock

  • 2.0 ml 1M Tris-HCl pH 6.8
  • 0.8 g SDS
  • 4.0 ml 100% glycerol
  • 0.4 ml 14.7 M β-mercaptoethanol
  • 1.0 ml 0.5 M EDTA
  • 8 mg bromophenol Blue

Final concentrations (1x)

  • 50 mM Tris-HCl pH 6.8
  • 2% SDS
  • 10% glycerol
  • 1% β-mercaptoethanol
  • 12.5 mM EDTA
  • 0.02 % bromophenol blue


Use of loading buffer

Dilute protein sample 1:3 into 4X sample loading buffer. Load on acrylimide gel in SDS-PAGE buffer.


Safety

β-mercaptoethanol is a severe irritant and is readily absorbed through the skin. SDS is a respiratory irritant in solid form and a mask should be worn while weighing it.

References

Links

  • 5x SDS loading buffer [1]
  • SDS loading buffer, Lynn lab, Uni Kentucky [2]
  • Wikipedia: SDS-PAGE, SDS
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