I want to elute active protein from native gel and still retain activity. Can it be done? Any reference would be helpful.
You should do the procedure known as electroelution. BioRad was used to have the equipment for the procedure (not cheap though), but any good protein-specializing vendor may have it. The procedure is simple and with the proper equipment takes only less than an hour.
For some proteins you can check enzyme activista directly in the gel. You hace to run a native PAGE and then inmerse gel in a solucion containing the substrate, preferibly in a buffer with a pH in which you know the enzyme is active. In this way, where tour enzyme is a colores banda will appear. This is what se usually do for laccases (a kind of metaloproteins) and is call zymogram
Hi Ravinder,
Bio-Rad electro eluter (Model 491 Prep Cell and Mini Prep Cell) should work very well for your purpose (As Oleg mentioned) and I had good luck using this instruments in my PhD research. We used to elute matrix metalloproteases 2 and 9 using this instrument in protein's active form for gelatin zymography (as Katiuska mentioned). The only constraint is that if you do not have an access to the instrument then the buying option could be expensive.
You can devise your own electro eluter in the lab using some common available resources as I did before using Prep Cell. I am sharing this protocol with you
1. Run the native PAGE in pair parallely in the same electrophoresis apparatus (Use preparative large well comb to load max amount of sample to be eletrophoresed).
2. Stop the gel before any protein starts to elute.
3. Take one gel and stain it with Coomassie blue following the set protocol and identify the band of interest.
4. Depending upon the molecular wt of the protein of interest choose dialysis tubing of proper Mr. cut off (10 kDa or 30kDa).
2. Cut it into required size and boil with dd water for 3-5 min in presence of a pinch of sodium bicarbonate.
4. Wash thoroughly inside and outside of the tubing with running dd water and lock one side using Spectrum dialysis clip or you can tie a knot using surgical suture. Do not let it dry and keep in cold dd water.
5. Using stained coomassie gel as reference make closest possible excision of the region in unstained gel with a surgical blade and mince the gel using the same surgical blade on top of a glass gel plate and transfer carefully the whole minced gel material into the diyalysis tubing and pour adequate amount of running buffer (25 mM Tris, 192 mM glycine, pH 8.3) to fill the tube leaving few cm empty length to lock the other end using clip or knot.
6. Take the agarose gel electrophoresis tank (Owl or Biotek) and fill it with the same running buffer and place the dialysis tube containing the minced gel horizontally between two electrodes.
7. Start running the power supply at 100V constant current to electro elute the protein from the minced gel for 60 min at 2-4 deg C. Do not touch or disturb the tubing.
8. After 60 min change the polarity of the electrodes by reversing the ports connected to the power pack and perform electrophoreseis in the same condition for 2 min to maximize the elution of the protein due the nonspecific attachment in the inside wall of the diyalysis membrane.
9. Stop run and take the dialysis tubing out. Let it stand vertically to settle down the gel at the bottom and aspirate carefully the solution inside tubing that ideally contain the electro eluted protein.
10. Concentrate this protein solution using conventional protocol (Amicon ultra filtration or membrane centrifugation with proper Mr. cut off filters)
11. Recover the concentrated sample and dialyze it against suitable buffer to retain active conformation of the protein in its purified form before you go for further activity assay.
To have a detailed info for the Bio-Rad Prep cell follow the link
http://www.bio-rad.com/en-us/product/model-491-prep-cell-mini-prep-cell
Good luck!!!
You can also add the substrate in the native gel . after then you can proceed for native PAGE. but its not possible for all the enzymes .
Hi,
I cut the band and sliced it. Elution was performed in an eppendorf in 2 volumes of 100 mM Tris-HCl pH 8,8 containing 5 mM of β-mercaptoéthanol ON at 4°C, the volume of buffer was twice the volume of the gel containing the band to be eluted.
Best
Hubert
Hi Hubert,
Your idea is good but using beta mercaptoethanol in the elution buffer may destroy the activity of the enzyme/protein. Because Ravinder is interested in eluting proteins ran on Native PAGE.
Regards,
@ Vikas,
Could you please elaborate the method you referred? It seems that you are talking something about running zymogram or some in gel activity assay....
Not sure. please come up with little extra info.
Best-
Please have a look at the detailed protocol attached to my response. There is also a description to produce your own electroelution equipment. In addition to Katiuska's comments, you may find hundreds of zymogram protocols. A classical reference is the book of Manchenko (1994).
Thanks everyone for valuable suggestions.. It was really helpful and I will definitely include your suggestions in my experiments..
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