Protein Protocols Protein Protocols

1006 Page and Thorpe
Fig. 1. SDS-PAGE minigel depicting purified IgG preparations derived from human serum
(lane 2), mouse ascitic flude (lanes 3 and 4), rabbit serum (lane 5), and sheep serum (lane 6).
Samples are elctrophoresed under reducing conditions and stained with Coomassie blue. All
IgGs consist predominantly of two bands comprising heavy (50,000 M r) and light (22,000 M r)
chains with no major contaminating proteins. Molecular-weight markers are shown in lanes 1
and 7 and their molecular weights (in thousands) given on the left.
3. Mix the sample in the ratio 2:1 with sample buffer.
4. Heat at 100°C for 2–4 min.
5. Prepare gel solution and running buffer as described in Subheading 2.
6. Assemble gel mold according to manufacturer’s instructions.
7. Add 30 µL TEMED to 10 mL of gel solution, pour this solution between the plates to fill
the gap completely, and insert the comb in the top of the mold (there is no stacking gel
with this system). Leave for 10 min for gel to polymerize.
8. Remove comb and clamp gel plates into the electrophoresis apparatus. Fill the anode and
cathode reservoirs with running buffer.
9. Load the sample(s) (30–50 µL/track), and run an IgG reference standard and/or mol-wt
markers in parallel.
10. Electrophorese at 150V for 1.5 h.
11. Remove gel from plates carefully, and stain with Coomassie blue R stain for 2 h (gently
rocking) or overnight (stationary) (see Note 3).
12. Pour off the stain, and rinse briefly in tap water.
13. Add excess destain to the gel. A piece of sponge added during destaining absorbs excess
stain. Leave until destaining is complete (usually overnight with gentle agitation).
4. Notes
1. The “minigel” is easily and quickly prepared consisting of a resolving (separating) gel only
and takes approx 1.5 h to run once set up. The IgG sample is prepared for electrophoresis
under reduced conditions, and is run in parallel with either a reference IgG preparation or
with standard mol-wt markers. The heavy chains have a characteristic relative molecular
weight of approx 50,000 and the light-chains a molecular weight of 22,000 (Fig. 1).