8 replies to this topic

[schaapje_86]

Dear all,

I am trying to optimize a buffer for a protein assay. In the orginal buffer is Tris, glycerol, DTT, NaCl, EDTA and MgCl2.
I am wondering what the function of MgCl2 is in my buffer? I read somewhere that it helps protein-protein interactions, but there is not much to find about this.
Does it form a complex with EDTA? and if so, what would be the purpose of that in my protein buffer?
Another thing to mention is that my proteins have a His-tag. Would EDTA have an effect on this?

I hope you can help, thanks!

[FlicFlak]

Hey, i was actually wondering this myself, as i have the same thing, although my MgCl2 concentration is extremely low.
Also, just in case, if you are doing a protein assay, like Lowry, the EDTA in the buffer will interfer with the result!! You'll have to do a precipitation step with TCA and resuspend in water or something to get rid of the EDTA.
Im sure you know this, but i got caught and took a while to figure it out!!!

[schaapje_86]

It's a fluorescent protein based assay (FRET) so it's a little bit different for me, some components have a detrimental effect on my fluorescence signal like NaCl but that's due to the nature of my protein-protein interaction. However, I couldn't figure out what function MgCl2 would have besides being another salt in my buffer..

[phage434]

Having both in a single buffer makes little sense.  The EDTA removes magnesium ions, the MgCl2 adds them.  You might want to watch out for using an EDTA containing buffer with His tagged proteins.  The EDTA will chelate out the cobalt or nickel in the column used for His tag binding.

[mdfenko]

due to disassociation constants, edta-mg will allow a trace of free mg in the solution. you are, in essence, buffering the metal ion.

[phage434]

Good point.  But I thought the constants were such that very little free Mg++ would be present.

[Astilius]

If there's any calcium in the sample mix then this will preferentially form an EDTA-Ca complex ousting the Mg and releasing previously bound Mg++ ions into solution.
Also, EDTA chelates Mg on 1:1 molar ratio so it will only mop up all the free Mg (to where the dissociation constant allows) if there's at least the same molar amount in there.

[schaapje_86]

Thank you all for your input. However, I am still not sure what the MgCl2 would do to my (fluorescent fused) proteins? Does anybody know this?

and Phage424, I don't use EDTA in my purification buffer so that's ok. Would have EDTA have any additional effect in my buffer here accept for acting as anti-microbal agent?

[Smog187]

In order to know whether or not MgCl2 has any effect on your protein, you should do FRET under low and High MgCl2 and see if you see a difference in signaling. We have MgCl2 in our buffers and it doesn't have any detrimental effects. We use MgCl2 to mimic ionic interactions that may occur. EDTA is not an anti-microbial agent