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Study Notes: The Laemmli Gel

Print out a copy of these Study Notes for future reference. This includes a long list of recipes that will form an important part of the rest of this section. Note that ‘tricks of the trade’ are given in italics.

The Laemmli Gel is the gel of choice for SDS-PAGE. Since 1970 there have been many variations on the original method but the overall system remains the same.

The Laemmli gel has the following features:

  • It is discontinuous with a
    • Stacking gel (3% acrylamide, pH 6.8)
    • Separating gel (5% - 20% acrylamide, pH 8.8).
  • The gels contain chloride as the mobile anion.
  • The upper and lower tank buffers contain glycine as the mobile anion and are at pH 8.8.
  • Sample wells are formed into the stacking gel and the sample (in sample buffer) is loaded into these wells.
  • An electric field is applied and the proteins (which are negatively charged due to SDS) migrate through the gel towards the bottom of the gel.
  • The stacking gel stacks the protein molecules into tight bands according to their Mr and the bands are then separated as they migrate though the separating gel.
  • Depending on the strength of the gel, smaller proteins may migrate off the bottom of the gel before the larger proteins at the top are sufficiently separated. This is not a problem if you are interested only in the larger proteins.

SDS-PAGE uses reasonably standard recipes for each of the components of the gel system. A number of stock solutions are made up. These are then used to make up the gel components.
Note: All reagents for the gels are of high purity but the reagents for the running buffers in the tanks are a lower (buffer quality) purity.

The stock solutions are as follows:

Stock Acrylamide Solution (30% w/v acrylamide, 0.8% w/v bis)
(Acrylamide is a potent neurotoxin and is a known carcinogen - treat with great care)
60.0 g acrylamide
1.6 g bisacrylamide
Make up to 200 mL in distilled water

1 M Tris Buffer - pH 8.8
(Will need to add almost all the water, then pH with HCl and adjust to 200 mL)
24.22 g Trizma Base (High purity TRIS)
Make up to 200 mL in distilled water
Adjust to pH 8.8 with HCl

0.5 M Tris Buffer - pH 6.8
12.11 g Trizma Base
Make up to 200 mL in distilled water
Adjust to pH 6.8 with HCl

10% w/v SDS
(Wear mask, careful of fine powder when weighing)
(Careful with mixing - generates strong foam)

10 g SDS
Make up to 100 mL in distilled water

10% w/v Ammonium Persulfate
(Make fresh each week)
0.1 g ammonium persulfate
Make up to 1.0 mL in distilled water

Stacking gel Recipe (3% acrylamide, pH 6.8)
(Add all but SDS and persulfate and degas then add SDS and persulfate - pour immediately)
1.00 mL stock acrylamide solution
2.50 mL 0.05 M Tris Buffer - pH 6.8
5 µL TEMED (polymerising agent)
6.37 mL distilled water
100 µL 10% SDS
100 µL ammonium persulfate (polymerising agent)

Separating Gel Recipe (say 10% acrylamide)
(Add all but SDS and persulfate and degas then add SDS and persulfate - pour immediately)

(For other strength gels the amounts of stock acrylamide solution and distilled water are adjusted accordingly - eg for an 8% gel the volumes used would be 8.00 mL and 10.35 mL respectively)

10.00 mL stock acrylamide solution
11.25 mL 1 M Tris Buffer - pH 8.8
8.35 mL distilled water
300 µL 10% SDS
150 µL ammonium persulfate

Note: The above volumes for the stacking and separating gels are based on the Bio-Rad SDS-PAGE gel, which has dimensions of 15 cm x 15 cm and a 2 mm thick spacer. In gel systems with other dimensions the volumes would need to be adjusted accordingly.

Stock Sample Buffer
(Make fresh each day)
25 µL 0.5 M Tris-HCl, pH 6.8
500 µL 0.1% w/v Bromophenol Blue
500 µL distilled Water
1000 µL glycerol

Stock 10 % w/v DTT Buffer
100 µL 0.5 M Tris Buffer, ph 6.8
10 mg DTT

Sample Buffer
(Use immediately to prepare samples, discard left over buffer)
40 µL stock sample buffer
40 µL stock 10% w/v DTT Buffer

Upper and Lower Tank Running Buffer - 10 x Concentrate
(Careful with mixing - SDS foams strongly)
60.7 g Tris (Buffer grade)
287.3 g glycine
20 g SDS (Buffer grade)
Make up to 2 L in distilled water

Upper and Lower Tank Running Buffer
100 mL 10 x Concentrate
900 mL distilled water
Add 450 mL to lower tank and 550 mL to upper tank.

Pouring the Gel
To pour the gel the appropriate ingredients are carefully aliquoted, mixed (note that TEMED and Ammonium Persulfate are added after degassing), degassed and poured into the gel apparatus. The gel should be left undisturbed under a layer of water to polymerise for at least 20 minutes. Remember that separating gel is poured first, followed by the stacking gel, and that the stacking gel has a gel comb inserted in it to form the sample wells.

Remove the sample comb carefully after 20 minutes, rinse the sample wells with tank running buffer and leave the buffer in them. Do not let the top of the gel dry out.

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