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Study Notes: Gradient Gels

Gradient gels have a higher concentration of acrylamide at the bottom than at the top and have a continuous decreasing concentration of acrylamide gradient from bottom to the top. Gradient gels are used for the:

  • determination of protein molecular weight (Mr)
  • separation of molecules that co-migrate on uniform gels.

Casting of gradient gels requires a gradient forming device (see below) and this is a more labour intensive practice than casting a uniform gel. Essentially two acrylamide solutions are made up with a high strength and a low strength of acrylamide. For instance, to cast a 5 - 20% acrylamide gradient gel, 5% and 20% acrylamide solutions are made up.Skip flash movie

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Notice that as the higher % solution drains from cylinder A, it is replaced by lower % solution from cylinder B. This has the effect of progressively reducing the % of solution added to the gel plates.

The casting device starts by layering a 20% acrylamide into the bottom of the gel but as this flows into the gel the 5% solution flows into the 20% container and starts to dilute it. The end result is a gel with 20% at the bottom and a 5% at the top with a smooth gradient in between.

To overcome the inconvenience of doing this yourself, pre-cast gradient gels are available from laboratory supply companies (in fact most types of acrylamide slab gels are now commercially available).

Gradient gels can be selected to suit the separation of particular ranges of size (the unit of Mr is kilodaltons or kd). The following table shows some examples.

Gel % acrylamide
Protein Size Range ( kd)
5 -15
20 - 200
5 - 20
10 - 200
8 - 15
10 - 100
8 - 20
8 - 150
10 - 20
6 - 150

To have an appreciation of what these protein sizes mean, a number of common proteins and their Mr are listed in the table below.

Name of Protein
Size in kd
Bovine Serum Albumin (BSA)
67
Heavy chain of immunoglobulin G
50
Ovalbumin
43
Alpha-chymotrypsinogen
25.7
Beta-lactoglobulin
18.4
Lysozyme
14.3
Cytochrome C
12.3
Bovine Trypsin Inhibitor
6.2
Insulin
3.0
Bacitracin
1.4

 

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