Electro Elution of my protein after SDS-PAGE - (Nov/28/2007 )
as I had massive problems to purify my protein I'd like to get it directly out of my SDS-gel after His6 purification.
To do so I would cut out the appropriate protein band (negative stain or stain a piece of the gel that can be used as a template). After it I think a good method would be electro elution (as I did already for DNA). Because we don't have a machine for it (...as it is found of BioRad i.e.) I'd put the gel piece in the right orientation into a dialysis membrane and electro elute the protein into the ambiance buffer using either a protein gel chamber or a dna separation chamber.
To get rid of SDS I'd dialyse against a sds free solution for several times. Protein could be concentrated using a concentrator (vivaspin) or a TCA precipitation.
The SDS content can be checked using a method after Hayashi et al.
To do the wholesome I found a few protocols:
- any suggestions what I have to look for using the separation chamber? are there problems regarding the resistor because its only a piece of the whole gel?
- to get rid of SDS, is it enough to dialyse the sample?
- do you have any other suggestions how to prepare a protein out of a SDS gel?
thank you for your help!
you can perform electroelution, as you suggest (and you seem to know how to set it up-you can check at the websites of companies that make electroeluter chambers for ideas on how to use an eppendorf tube as an electroelution tube).
or you can homogenize the gel slices in buffer (as detailed in the document from pierce).
when you remove sds, you need to use more than just a sds-free buffer to remove the sds from the protein. triton x-100 or nonidet p-40 is required to displace the sds (and, maybe, renature the protein).
great, thanks for your reply mdfenko!
The document of pierce describes for homogenization an overnight incubation step at 30 °C. Do you think the temperature could do harm to the protein? Would you place in the buffer a choice of protease inhibitors? (....as you never know whether there still are some in the gel slice or not AND if the SDS-PAGE did not destroy its activity).
since the gel slice should represent a purified protein i wouldn't worry about proteases.
the protein is already denatured so prolonged exposure to 30C should have no significantly adverse effect.
For the removal of SDS there exist also a whole bunch of protocols. You already mentioned the way using detergents like Triton X-100 and NP-40.
There is a protocol http://www.bio.net/bionet/mm/proteins/2000-July/008439.html where a small percentage of sucrose is used to solubilize the protein in a cold acetone surrounding. It says that SDS is precipitated by acetone and you do not have the adjacent problems of detergent in the protein sample. Do you think this is a possible sideway?
what about conventional methods like TCA precipitation or chloroform/methanole precipitation? Does it only precipitate proteins and remove sligthly bound substances but not SDS?
if you looked at the rest of that thread you would have seen that there was some controversy about whether acetone would work.
the triton method works and afterwards you can remove the triton from the buffer (eg-with a detergent column).
tca and chloroform/methanol denature and precipitate the protein but don't remove sds.
1) Electro elution
worked very well. The protein eluted into the surrounding buffer. If somebody needs a more detailled protocol I can advise this publication: "Electroelution as a simple and fast protein purification method: isolation of an extracellular Xylanase fromo Bacillus sp. CCMI 966" by Sá-Pereira P and Costa-Ferreira M (1999, Enzyme and Microbial Technology).
2) Crushing the gel pieces
is an ungracious job. For crushing I used a glass stick but it does not really work. You get pieces but its more like a slimy matrix (even if you freeze your gel). After centrifugation (13'000 rpm - 40'000 rpm) the gel does not sink but you get a mix between gel and fluid. Only a bit swims on the gel-slime. I can not recommend that way, although the effort is less than choice 1).
To purify a protein the gel elution method really is a great way when its hard to separate the desired protein via chromatography. But keep in mind that you have to remove the sds from the resulting sample!
since electroelution works well for you then you should continue using that method, but...
the homogenization method works better than you found. we use a homogenizer to smash the gel (teflon-glass tissue homogenizer or pellet pestle with an eppendorf tube and using a motor to spin the pestle). besides the ability to centrifuge to separate the gel from the solution, you can also pour into a column (or spin column or scintered glass funnel) to flow the eluted protein away from the gel.