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Methods:Tris-Glycine PAGE

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Contents

Purpose

Tris-Glycine (aka Laemmli) gels[1] are commonly used to resolve or purify proteins denatured by Sodium Dodecyl Sulfate (SDS). Tris-Glycine gels use a stacking gel to compress the sample into a narrow band before it enters the resolving gel. This leads to much sharper bands than would be seen in gels lacking a stacking gel. Tris-Glycine gels can resolve proteins by size. Very small proteins and peptides do not resolve due to interference from the glycine/pH discontinuity front; Tris-Tricine gels are preferable for low MW proteins.

Materials Needed

Warning.png

Acrylamide at the concentratons used for laboratory gel electrophoresis is a hazardous chemical. Acrylamide is aborbed through the skin and is a cumulative neurotoxin. Always wear gloves when handling acrylamide and acrylamide solutions, and clean up any spills immediately. It is often preferable to purchase premade acrylamide solutions to reduce hazards due to acrylamide dust. See MSDS

  • H2O
  • 30% acrylamide:bis-acrylamide stock solution
  • 1M Tris (pH6.8) for the stacking gel
  • 1.5M Tris (pH8.8)(mL) for the resolving gel
  • 10% Ammonium persulfate (mL) - make fresh
  • 10% SDS
  • TEMED ()
  • Water saturated butanol
  • Running buffer:
    Make from 10X Stock
    0.25 M Tris pH 8.4 – 8.9
    1.92 M Glycine
    1% SDS

Protocol

Wear gloves and work on disposable absorbent paper to catch any spills.

  • Assemble the gel apparatus. Use a marker to mark the glass to the desired height of the resolving gel.
  • SDS PAGE calculator from Chang Bioscience can be used to calculate volumes needed. Enter the final %acrylamide and desired volume. Mix the gel solutions without the APS and TEMED. Degas them before use. Gels will usually polymerize without being degassed, but oxygen inhibits the polymerization reaction, and the time will be more consistent if you degas first.

Pour the resolving gel

  • Add the APS and TEMED to the resolving gel solution and quickly pipet the solution into the gel slab or tube to the desired height. Immediately layer water saturated butanol onto the gel - this prevents formation of a curved meniscus and gives a flat surface to the top of the gel.
  • Wait for the resolving gel to polymerize completely. Usually you can monitor this by watching the polymerization of the leftover resolving gel solution.
  • Pour off the butanol and rinse the top of the resolving gel with water.

You can store the resolving gels for later use as long as you keep them wet.

Pour the stacking gel

  • Add the APS and TEMED to the resolving gel solution and quickly pipet the solution into the gel slab or tube.
  • If using a slab gel, insert the slot former. For tube gels, layer water-saturated butanol to give a flat surface for loading.

Links

User notes

References

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  1. Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680-5 PubMed EcoliWiki page