I have a project to isolate a protein with biological properties from a plant. The purified protein forms four bands with similar molecular weight on SDS-PAGE (30–35 kDa) in the presence of 5 % 2-mercaptoethanol, and two bands with lower MW (27–30 kDa) when 2-mercaptoethanol is omitted.

How can these results be explained? What is the easiest and lowest cost method to separate closely related proteins (if that’s what these are, and not just subunits of a single protein)?

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    $\begingroup$ I have made edits to try and make this question more readable. Please check my edits to ensure I have not changed your intended meaning. $\endgroup$ – user137 Jun 20 '16 at 10:45
  • $\begingroup$ What do you mean by "purified protein"? Are you asking if your protein is actually purified, or are you confident that it is pure and want to know how to handle next steps? $\endgroup$ – iayork Jun 20 '16 at 12:07

2-mercaptoethanol (2ME) reduces the disulphide bonds in proteins. If disulphide bonds are connecting two polypeptide chains ("intermolecular") then 2ME would cause them to separate and therefore instead of a higher molecular weight (MW) band you would get one or more lower MW bands.

However, if there are disulphide bonds in the same polypeptide chain, they may cause the chain to fold up. This results in the polypeptide chain to effectively have a smaller hydrodynamic radius compared to the fully denatured chain. In such cases reducing the disulphide bonds using 2ME would cause the band to shift to a higher MW.

What is the easiest and lowest cost method to separate closely related proteins

This is a different question altogether and there are several techniques depending on how "dissimilar" or "similar" the closely related proteins are. 2D PAGE (with isoelectric focussing) is one technique that can offer higher resolution compared to a normal SDS-PAGE. There are many other chromatographic methods.

  • $\begingroup$ @R.Rasoulpour iayork's comment is under the question. Anyway, if you think this answer addressed your problem, please consider accepting it by clicking on the check mark/tick to the left of the answer, turning it green. This marks the question as resolved to your satisfaction, and awards reputation both to you and the person who answered. $\endgroup$ – WYSIWYG Jun 20 '16 at 17:37
  • $\begingroup$ this is the right answer $\endgroup$ – rhill45 Apr 11 '17 at 4:24

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