3
$\begingroup$

In sample buffer preparation we add EDTA, but if SDS-PAGE is for protein then is it necessary to add EDTA in sample buffer? What is role of EDTA in sample buffer for protein separation for SDS-PAGE.

$\endgroup$
  • $\begingroup$ In my opinion EDTA is dispensable. Common proteases like serine proteases do not depend on metal ions. As the post in this page suggests, there are EDTA free loading buffers. This discussion in researchgate suggests that EDTA is excluded from protease inhibitor cocktails because it may interfere in downstream steps. I am commenting because I am not able to verify the dispensability of EDTA, from any reliable source. $\endgroup$ – WYSIWYG Jan 7 '15 at 12:27
  • $\begingroup$ I've never used EDTA in sample buffer. The combination of SDS and DTT (or β-ME), along with the boiling step, is enough to completely denature and inactivate pretty much every enzyme, including proteases. $\endgroup$ – MattDMo Jan 7 '15 at 16:24
3
$\begingroup$

EDTA is a complexing agent for bivalent cations and is usually used to inhibit enzymes (proteases, nucleases) which need these ions in their active center to work. Without it they do not attack the sample.

The presence of EDTA in the sample buffer is interesting, since Laemmli's original paper from 1970 ("Cleavage of structural proteins during the assembly of the head of bacteriophage T4.") doesn't use it and I think it is not necessary.

The reason for this is simple: Protein samples are usually generated in buffers which contain different protease inhibitors, which protects the samples. Additionally the purpose of the sample buffer is to denaturate (at least for the standard SDS gels) the protein sample completely, so it will run uniformly. Both steps ensure that proteases are either inhibited or completely denaturated, which will both protect the sample.

The only reason I can think of for using EDTA is that it can help to destabilize your target protein, when a co-factor which is important for the structure is complexed by EDTA and thus keep it in solution after the denaturation.

There are of course ressources which state that EDTA is necessary for protein integrity, but they only say it is there to inhibit proteases by complexing metal ions (see here for example).

As a 2x sample buffer, I use the following recipe (this can also be made 5x if necessary), which contains EDTA, but can also be done without. If you prefer β-Mercaptoethanol, you can use it in the same concentration as the DTT (which has the advantage of being less smelly):

2x sample buffer:

  • 4% SDS
  • 20% glycerol
  • 2 mM EDTA (ethylene diamine tetraacetic acid) - can be made without
  • 100 mM Tris-Cl, pH 6.8
  • 200 mM DTT (dithiothreitol)
  • 0.1 % (w/v) bromphenol blue dye
$\endgroup$
  • $\begingroup$ AFAIK only metalloproteases use metal ions (Zn and Co); these are not the most common proteases that one would encounter. $\endgroup$ – WYSIWYG Jan 7 '15 at 11:56
  • $\begingroup$ There are some calcium dependent proteases. You may also want to inhibit kinases or phosphatases. $\endgroup$ – Chris Jan 7 '15 at 12:14
  • 1
    $\begingroup$ There are very few of Ca dependent proteases. I am not sure kinases need metals as cofactors (activation is a different thing; that's signaling). Alkaline phosphatase needs Zn. But I guess this is not as common as the dependence of nucleases on metal ions. In any case we do use a protein inhibitor cocktail while doing extraction. Loading shouldn't require EDTA; you are just denaturing. $\endgroup$ – WYSIWYG Jan 7 '15 at 12:26
  • $\begingroup$ @WYSIWYG I looked into Laemmli's paper from 1970, and he doesn't mention EDTA at all. My guess for the moment is, that it has been added later for unknown reasons. I think since the concentration is not high anyways (1mM final), there will be no harm if it is removed. I'll check that later and edit the answer. $\endgroup$ – Chris Jan 7 '15 at 12:45
  • $\begingroup$ Yeah I guess it is like one of those occult biolab practices of putting every coloured substance including copper sulphate solutions, covered in aluminium foil at 4⁰. Not sure though. $\endgroup$ – WYSIWYG Jan 7 '15 at 12:47
3
$\begingroup$

I agree with the other comments but want to add something. EDTA works as a stabiliser for bME. If you have bME in your 5x sample buffer and want to store it for a long time period add EDTA, otherwise you have a big loss of activity. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/m7154pis.pdf

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.