Elimination of the polyethylenimine - (Mar/24/2009 )

I am interested in eliminate the PEI after a clarified extract treatment with this polimer in order to get rid off the DNA, I know that after precipitation i still have a little quantity of PEI for two reasons:
1-The pH still is very high (aprox. 9)
2-When i use a Ni2+ resin after this treatment i loose the Ni2+ of the resin (it turns white from blue) due to PEI can effectively complex heavy metal ions.
However, i guess that in my purification procedure i am eliminating any protein that binds DNA and is precipitated with PEI because when i got out this step from my purification procedure, i obtained several false positives in the ELISA assays performed with this recombinant antigen, therefore I need this step or a similar one.
I dont know if this polimer is easily dialysed, if anybody has a clue.
Other option is maybe use a heparine resin which get rid off the proteins which are able to bind DNA, the fact is that i don´t want to change very much the purification procedure and PEI is much inexpensive than heparin.

Any of you have an idea about dialysis of PEI or maybe using a desalting column with the clarified extract after PEI precipitation.



Thank you in advance

Hello,

I use PEI regularly and have never faced this problem. The final concentration is 0.1 % and yes I do dialyse the PEI stock.

Here is the protocol:

4 g of PEI was weighed and was dissolved in approximately 10 ml of 25 mM Tris-HCl buffer (pH: 8.0). This solution was then transferred to a dialysis tubing (MWCO 3,500 Da) and dialyzed against 25 mM Tris-HCl buffer (pH: 8.0) for at least 24 hrs. The PEI solution was then recovered from the tubing and measured. Percentage concentration (w/v) was calculated and the solution was stored at 4 degree celcius.

I get a feeling that the final concentration that u r using is higher than 0.1% or yr PEI stock has Impurities.

ALternatively, you can avoid PEI and use DNAse.

Hope it helps.

Best,
TC

Thank you TC,
Then i will try the dialysis. The fact is that i don´t want to use DNAse because i want to eliminate some proteins that copuld be binded to the DNA, the PEI is precipitated and therefore i get rid off this protein in the centrifugation, but the DNAse only degrades the DNA. Firstly, I perform a meassure of the DO at 260 nm of my crude extract and i ususally use a relation 0.25% PEI per 120 OD 260, which normally for an E. coli extract is aprox. 0.4 g for 50 ml of extract (i use a stock solution of 10% PEI). The, when the mixture is homogeneised, it is pelleted and the sob is used to continue the purification procedure. Then i will try to dyalise the extract before puting in contact with the Ni-resin.


Thank you for your prompt answer

Hey

My protocol is different from yours:

1. I can never choose what my stock percentage will be as it depends upon the final volume I get after dialysis.

2. I don't measure the OD, I just measure the volume of the lysate and add 0.1 % using %1V1= %2V2.

3. The required volume of PEI is added dropwise along the walls of the flask containing teh lysate. Also, teh flask is swirled while the addition is ona nd is on ice. If it is added directly and w/o swirling, the local conc. of PEI increases and leads to target protein precipitation.

4. This I pellet and use the supernatent.

Hope it helps.

Best,
TC

paramyosin on Mar 24 2009, 04:09 PM said:

Points 3 and 4 are the same for me. But, i have never prepared the PEI stock solution like that, i have a sigma stock 50% of PEI and i maintain a stock 10% at 4ºC to add to my lysated and the quantity i use depends on the meassure i have told you. My idea is to dialyse the lysed after precipitation to eliminate the PEI which didn´t have pelleted and i guess you were meaning that you dialyse the PEI to perform your initial stock. In any case, i think that both possibilities are the same regarding the capacity of the PEI to be dialysed. didn´t you notice the presence of PEI after the precipitation, when you recover your supernatant?

Hey

How do you notice PEI? The colour of the beads never changed to white, the lysate was always clear and I never observed anything unusual.

I think the 0.1% PEI by volume that I use if much lower than the 0.25% that you use by OD measurement. Try decreasing this on a small aliquot of yr lysate (try different conc. and use a bench top centrifuge).

The advantage that I see with dialysis of stock PEI is that yr lysate won't be seeing any other additives that the PEI stock would have (if any).

Also, dialysis before addition will take care of any monomers that would be present in the PEI solution....so they won't react with yr lysate.

That is what I think but I may be wrong

U can try this too: in one case add dialysed PEI and in another case go without dialysis and see the difference.

Best,
TC

paramyosin on Mar 24 2009, 06:00 PM said:

I will try. Thank you a lot for your ideas

you can't dialyze out the PEI from your sample, cause it's way too large and too hydrophobic

the usual protocol is to do a (NH4)2SO4 precipitation after your PEI precipitation

so you just take your supernatant from your PEI precipitation and add ammonia sulphate to precipitate your protein. You pellet your (NH4)2SO4-protein precipitates via centrifugation, redissolve them in buffer and dialyze. Afterwards you can go ahead with your Ni column run.

Hi all,
how you measure PEI concentration in prepared solution?
Thanks

Hi all,

I am trying to purify the RNAP. In the protocol I am using they do PEI precipitation but in my case I want the pellet instead of the supernatant. Anyway my problem is I have to prepare PEI 10% (v/v) and I have PEI 50% (w/v). To prepare your stock at 10% (v/v), do you dilute the concentrate 5 times? I know is a silly question but the strange for me is PEI is a solid and you have to prepare it in %v/v.

Thanks i advance.