Dear all,
I'm looking for Cpf1 purification protocol and I found a protocol in which the authors added GABA to the buffers ( from equilibration till the eluition). I'd like to know if I can substitute with something different.
Thank you in advance
Hi Alessandra Bellan,
I would like to analyse that protocol. Could you please provide some source information or pdf file of that research paper.
Thank you.
The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies.
Article Expression, purification and structural analysis of function...
Article Expression and purification of a functional heteromeric GABA...
Ah, so you want to purify a GABA receptor! Then it makes sense to add its ligand during purification to stabilise it. This is particularly true for an oligomeric protein, that may fall apart w/o ligand. Remember that the purification of membrane proteins usually requires solubilisation, that is, the transfer from the membrane environment into detergent/lipid mixed micelles, a rather harsh procedure.
The GABA could, in principle, be replaced by anything that binds to the GABA binding site with sufficient affinity.
It would be good to have a look at the protocol.
GABA is a polar zwitterion at low pH and the reason they might put it into the equation are different. Maybe it is used to stabilise the Cpf1 conformation (or to adjust the pH for structural stability), but i believe there are better alternatives. It's a quite small molecule, so it might be used to bind to some other enzymes and proteins to increase their molecular weight and help the purification resolution (i'm still guessing here). Or maybe its size is so small (ish) and you might want to know if it somehow interacts with the endonucleae itself (but i highly doubt so).
Honestly if you were to purify a GABA receptor or transporter (like Engelbert Buxbaum or Asif Shahriar are suggesting) yes, that's the reason why you want the ligand in it.
Otherwise i see very poor connections on using GABA on an endonuclease :\
Keep us posted!
From my own perspective you could replace it:
I think GABA is employed in making buffers generally speaking such as gaba/acetic acid Buffer with pH (4-5).
here are some protocol sources:
http://m.cshprotocols.cshlp.org/content/2006/1/pdb.rec10508.full?text_only=true
https://mtc-usa.com/celerity_5.asp
and
also as mentioned by others its a zwitterionic molecule, so with amino acids, it helps in achieving a chemical equilibrium that maybe is essential for maintaining protein conformation as in providing a stabilizing effect.
Proceeding from there you could substitute it, also check this Cpf1 purification procedure for more information:
https://bio-protocol.org/e2842
Good-luck!
Ahmed Hablass i agree... I just assumed there were cheaper and easier ways to stabilise pH :D
Thank you!
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