I have done quite a lot it in a very simple way. The cut fragments of the gels were put in dialysis bag filled with buffer I (200 mM Tris-acetate pH 7.4, 1% SDS, 1.5% DTT) and put into apparatus for horizontal electrophoresis filled with buffer II (50 mM Tris-acetate pH 7.4, 0.1% SDS) (crosswise to the electricity flow) and 100 mA were applied for 4-5 hours in a cold room. Then dialysis bag was transferred into buffer III (20 mM Tris-HCl pH 6.8, 0.02% SDS), about 5 liters of it and dialysed overnight in a cold room. Then the buffer with proteins from dialysis bag was transferred into Eppi vials and precipitated with a cold acetone (4-5 volume of cold acetone per 1 volume of protein mix) and precipitated in -20 overnight. Then centrifuged, protein pellet was washed with cold acetone, centrifuged once again, dried (carefully as to not overdry it) and dissolved in PBS.

What was important for an efficient electroelution was that that the bands of the gel were cut out from the poliacrylamide gels stained for 10 min only with water Coomassie (0.05% Coomassie solution in water) and destained by washing a few times with water - the staining is very delicate, wanishes quickly so You need to cut out the right bands fast using a scalpel. To get a lot of protein I have put my cut out fragments into -80 freezer and after I got enough of them I made an electroelution.

By electroelution, you have your protein in 400 µl buffer

I think this link should answer your question :

http://www.researchgate.net/post/How_to_elute_a_protein_band_of_interest_from_SDS-PAGE_or_NATIVE-PAGE_gel

Fung: There are various protocols out there to address your issue. Here is one of the most recent ones:

http://www.ncbi.nlm.nih.gov/pubmed/23512288

"Mass spectrometric peptide analysis of 2DE-separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation."

Agree, electroelution may work. I did this. Works well for my leech homoeobox protein isolated from e. coli lysates.

I have done quite a lot it in a very simple way. The cut fragments of the gels were put in dialysis bag filled with buffer I (200 mM Tris-acetate pH 7.4, 1% SDS, 1.5% DTT) and put into apparatus for horizontal electrophoresis filled with buffer II (50 mM Tris-acetate pH 7.4, 0.1% SDS) (crosswise to the electricity flow) and 100 mA were applied for 4-5 hours in a cold room. Then dialysis bag was transferred into buffer III (20 mM Tris-HCl pH 6.8, 0.02% SDS), about 5 liters of it and dialysed overnight in a cold room. Then the buffer with proteins from dialysis bag was transferred into Eppi vials and precipitated with a cold acetone (4-5 volume of cold acetone per 1 volume of protein mix) and precipitated in -20 overnight. Then centrifuged, protein pellet was washed with cold acetone, centrifuged once again, dried (carefully as to not overdry it) and dissolved in PBS.

What was important for an efficient electroelution was that that the bands of the gel were cut out from the poliacrylamide gels stained for 10 min only with water Coomassie (0.05% Coomassie solution in water) and destained by washing a few times with water - the staining is very delicate, wanishes quickly so You need to cut out the right bands fast using a scalpel. To get a lot of protein I have put my cut out fragments into -80 freezer and after I got enough of them I made an electroelution.

This might help...

electro-elution is the way to go. To add to what Patricjya said, make sure to reverse the electrodes at the end just for 1-2 minutes to extract protein stuck to the dialysis membrane

you have got perfect answer from Patrycia, It should work.

K. Fung,

I agree with Patricjya. Electro-elution is the way to go. If possible, I would like recommend the BIORAD apparatus (Model 422). It should work very well.

Best,

Edward

The best way to do it is to excise the gel-containing your protein of interest and put in a bag with appropriate buffer. run the horizontal electrophoresis. you will be able to elute your protein by that way. the same method is used to elute DNA fragment from agarose gel. Thanks. Simon

I have done it couple of times following the similar method in preparation for the Mass-Spec analysis to know exactly how many proteins are interacting with the protein/s of my interest and how many peptides are there in each of these proteins. Hope it helps.

I have follwed the protocol you mentioned and works pretty good for me, especially because the enzyme activity I was purifying wasn't affected by the treatment. Electroelution is the way to go if you do not care in maintaining enzyme activity.

Thanks everyone for the reply! Unfortunately, we don't have electroelution instrument in our lab. I don't think so I can do this. I wonder if I could use Novex gel electrophoresis mini tank (vertical position, not horizontal one) for running the dialysis. Is anyone using microcentrifuge filter to elute the proteins?

We have used electroelution, and it certainly works, but considering the non-availability of equipment I am letting you know what we did before we acquired the equipment. Excise the gel piece with the band, then crush it in an Eppendorf tube in an elution/renaturation buffer (20mM Tris buffer, pH 7.6, containing 1% Triton-X, 1mM EDTA, 2mM DTT, 100mM NaCl). Incubate at 37C o/n. Shake for 4 h, centrifuge, dialyze supernatatnt in 10mM Tris buffer, pH 8. Check yield. The yield in our case was somewhat low, but we got what we wanted.

If biological activity is not an issue, renaturation is not required. Hence a simple buffer should suffice, however, we have no experience with it, since we needed to retain the biological activity and hence renaturation following SDS-PAGE was a must.

I read one paper by Li et al. published in Analytical Biochem 2012 vol 430; doi: 10.1016/j.ab.2012.07.008, where they developed a simpler monolithic column electroelution You may give it a try.

Try our elution and KDS precipitation method.

See:

Rossini K, Rizzi C, Sandri M, Bruson A, Carraro U.

High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins. Electrophoresis. 1995 Jan;16(1):101-4.

Carraro U, Doria D, Rizzi C, Sandri M.

A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions, and then allows recovery and quantitation of proteins. Biochem Biophys Res Commun. 1994 Apr 29;200(2):916-24.

Sandri M, Rizzi C, Catani C, Carraro U.

Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation. Anal Biochem. 1993 Aug 15;213(1):34-9.

maybe try this, ignoring the trypsin steps. http://www.lamondlab.com/pdf/LLingeldigestion.pdf

Hi Kokwai,

The method you describe should work well and simple to do.

This is a very routine technique. If your bands are stained with Coumassie blue then simply destain the strip of the PAG and depolymerize (information readily available even on the net) and elute the protein. But the cautionary note is, be careful if you are doing this from a native PAGE, where you may be interested in getting the active protein.

The electro elution works very well.

Slice the nondenaturing PAGE by smashing

and dissolving the gels in 2% (v/v) triethyl amine (TEA) in

distilled water for 1 hour. The smashed gels with TEA water

centrifuge to collect the supernatant and lyophilize

to get frozen-dried proteins. resuspend the Protein in 10 mM potassium phosphate buffer (pH 7.0) and dialyze overnight.

ref: Shoji S, Ichikawa M, Yamaoka T, Funakoshi T, Kubota Y. High-sensitivity

amino acid analysis of stained peptides and proteins from a sodium

dodecyl sulphate-polyacrylamide slab gel. J Chromatogr 1986;354:

463–70

I used the method described in this paper for the quantification of radioactivity: Anal Biochem. 1991 May 15;195(1):172-6. A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels. Springer WR. However, I am not sure if you would able to recover functional proteins, since the gel slices will be solubilized in a periodate solution.

I agree with Balaji Saadashivam. I have used it keeping the buffer solution chilled. I have recovered the protein from native PAGE and could record the activity too.

I've done it using electroelution in the D-tubes from novagen. These are small tubes with two dialysis membrane windows on the side. You stain the gel, excise the band cut it in rectangles and put inside the tube with buffer (Used TGS). The whole thing is put in an horizontal electrophoresis apparatus using a little plastic support they sell on the kit. Takes like 2h depending on protein size. In the end you just remove your gel fragments from the tube and you can dialyse directly this tube with the protein solution to get rid of SDS and other contaminants,

You can try with In-gel tryptic digestion kit. It's a quite simple process by using of trypsin and other alkylation reagents. Here is a link for you :

http://www.piercenet.com/browse.cfm?fldID=932A4A8B-9433-4A5E-A91D-7416CE9A89E7

We used to cut the band in pieces then force it through a needle to break it up. This might help if your protein is high molecular weight. Then put it in a dialysis bag in a horizontal gel.. The last thing we did was to reverse the polarity of the electrophoresis, briefly, which was supposed to release protein that was adhered or partial transferred through the dialysis membrane.

Analytical Biochemistry

Volume 109, Issue 1, 15 November 1980, Pages 76–86

Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: Results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes

Dayle A. Hager, Richard R. Burgess