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Does sample buffer require EDTA for protein separation on SDS PAGE?

Does sample buffer require EDTA for protein separation on SDS PAGE?


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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.


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

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


Watch the video: SDS PAGE principles - simple animated tutorial (May 2022).


Comments:

  1. Amado

    I agree, this is a great option.

  2. Jedaiah

    I'm sorry, but in my opinion, you are wrong. I propose to discuss it.

  3. Volker

    I confirm. And I have faced it. We can communicate on this theme.



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