I am using 1/5 volume of ice-cold PEG–NaCl solution : To 200 g of polyethylene glycol 6000 and 146.1 g of NaCl add distilled water

to 1 L, heat to dissolve, and autoclave. After 4oC incubation, Centrifuge at 10,000g for 20 min at 4°C. Twice longer incubation gave me 100-fold increase in p.f.u./ml.

Hey Hadrien,

I also do PEG precipitation to isolate my viruses. I use 'PEG Virus Precipitation Kit' from BioVision and always get good results.

Two major variables: the concentration of PEG and sodium chloride. Remember that if the concentration of your particles is very small, you will not get anything. The combination of PEG and sodium chloride is to neutralize the particles to become insoluble (precipitate). If the concentration is low, particles becomes insoluble but they do not aggregate (low concentration) to form large precipitates.

Both... Type of PEG and PEG concentration are important..you can try using 12% PEG 8000...It works quite well for me...

Hi,

I used to use the PEG-8000 40% to purify HCV as well as Dengue virus about once month. I attach the protocol here but it's really easy. Basically, you dissolve the PEG in PBS and mix this with your VLP-containing supernatant for a final concentration of PEG of 8%. Than incubate at 4C overnight. In the morning, spin down at 8000g for 15min and harvest the pellet with PBS. Usually, I filter this virus-containing PBS with syringe filter for more purity. But remember that, with the PEG, you will also precipitate all the proteins of the serum, the media, etc... I would recommend you (for more purity but not mandatory) to do a sucrose gradient to be sure that you keep your VLP clean but it's not an easy way...

Finally, we use as well the VLP in the lab and we produce them, we just concentrate them with Millipore column with a cut-off of 100kDa and then keep them with polybrene to increase the infectivity of your VLP, ONLY if they are retrovirus-based VLP.

Hope it helps.

Forgot the protocol...

Hi,

I think you can try by PEG 20 percent with NaCl 2.5 M. Its better to use 1/6 volume of mentioned solution.

You are working in a multidimensional room. The precipitation depends on temperature, ionic strength, pH, PEG MW and the virus/protein (mixture) itself. Thy to modify only one parameter per experiment.

First of all try to diminish the amount of volatiles in your mixture by vacuum evaporation or liophylization to enhance concentrarion, even if you already have added PEG to the mixture. While the concentration becomes high precipitation should occur in situ as some of our colleagues have clearly stated. Hope this helps.

I used for protein precipitation a final concentration of PEG 6.000 MW- of 6 to 12 %

in deionized water. 6 to 12 thousand rpm should be enough.

My simple procedure for PEG preoccupation of virus supernatants such as influenza in allantoic fluid or VSV culture supernatant is to dissolve 8 % PEG8000 (W/V) directly into the sample (previously clarified by 4 k rpm X 15 min) by shaking and then incubate at 4 C for 1 hr. Pellet at 7 k rpm for 20 min in a Sorval GSA type preparative rotor. Drain and resuspend in PBS or STE and do further purification for virus ore extract nucleic acid directly from the concentrate.

Have you tried Ferric Chloride floculation method? There is a protocol form Sullivan's lab

http://www.eebweb.arizona.edu/faculty/mbsulli/protocols/Ferric_Chloride_Precipitation-TMPL_v6.pdf

I agree with most comments in terms of MW and % but one variable I did not see addressed is the protein concentration of the solution where the viruses are. This is important, since a high concentration of proteins (as in serum) will help the precipitation process. So, in other words, try it out with the variables mentioned before, but the concentration procedure (and the efficiency) will depend on even suspended solids in your starting solution.

Dear Hadrien,

If you don't found an already published protocol for optimizing PEG precipitation, or if the protocol published is not specifically referred to the case of your protein, you should use current methods of experimental optimization based on statistical criteria. Some colleagues already have noticed you that precipitation depends on mutiple factors: temperature, ionic strength, pH, PEG MW and the virus/protein (mixture) itself among others. However, in contrary to the procedure to modify only one parameter per experiment you should use a COMPLETE OR FRACTIONAL FACTORIAL EXPERIMENTAL DESIGN to firstly identIfy which are the relevant variables, and thereafter a SECOND ORDER RESPONSE SURFACE DESIGN to characterize the best combination of variables that optimize your problem.

Best regards.

Daniel

PEG 6-8000 is OK. You can optimise the salt concentration and the centrifugation speed with a few experimental trials based on other references available in the literature for similar isolations

Here is a good historical treatise using to precipitate virus and proteins: Juckes IRM, Fractionation of proteins and viruses with polyethylene glycol. Biochimica et Biophysica Acta 229: 535-546, 1971.

I am using 1/5 volume of ice-cold PEG–NaCl solution : To 200 g of polyethylene glycol 6000 and 146.1 g of NaCl add distilled water

to 1 L, heat to dissolve, and autoclave. After 4oC incubation, Centrifuge at 10,000g for 20 min at 4°C. Twice longer incubation gave me 100-fold increase in p.f.u./ml.

In my opinion, before introducing any variables, it is important to select a standard procedure from a good manual and check whether you have followed it strictly after understanding every step in detail. A small deviation from the procedure can result in failure.

Fresh Solution of 40%PEG8000(or plus 2.5M NaCl) are definitely demanded. I used old PEG solution(4 month old) and perform as before, and I did not get any PEG-virus pellet in bottom after 3000g  spin.

Ms Rupali, can I have the reference for you protocol Plz.

This is an old thread but I some questions. I am ligating some adapters to a smear of DNA (ranging from 100bp to 30 kb). Will it be possible to selectively remove unligated adapters (25bp and 50bp) from a ligation mix using a particular % of PEG and NaCl/MgCl2? The concentration of linkers will be high in the ligation mix in order to achieve a particularly tough ligation. So will be the amount of ligase enzyme. I have tried some spin columns like Qiagen PCR purification, Zymo's select a size kit, NEB's Monarch etc. All of these kits do remove unligated Adapters, but all of them leads to a heavy loss of DNA with ~only 40-50 % recovery. One more fact: the adapters are biotinylated so that specifically the ligated DNA can be pulled out later with streptavidin beads. 

I would like to thank the repliers and contribute some results. I tried to precipitate the retrovirus I produced. The virus structure was composed of Gag and Env from MMLV.

I tried to use all the combinations of the following choice (PEG6000 or PEG 8000) , (20% stock concentration or 40%), (2.5M NaCl in stock or not)

The solution PEG6000 - 40% concentration - no salt did precipitate the virus into a nice pellet after 4 celsius degree overnight incubation (1:4 PEG to virus supernatant v/v) and 10000g 20 min centrifuge.

The solution PEG 8000 - 40% concentration - no salt precipitated the virus into a pellet whose boundary was not smooth. 

All other solutions did not work for me.

Hi, Ye Tian!

How do you know that heterologously-produced virus-like particles were produced? 

Need to check it preliminary, there are several methods to do it.

Actually, PEG (salt helps) is able to precipitate almost all cellular and viral polymers,cause it binds water. You just need to find  its (and salt) optimal concentations. Sometimes the LiCl addition might be efficient as do many water-binding  agents.  

let me know how to do PEG fractionation of partially purified protein.

Total procedure

I don't knwo how important is for you this step of precipitation. In any case, if were important you should read the original articles, or at least some of them close to related-type of substance and matrix support that you have to work (it could be important what other protein contaminants you can precipitate together with you molecules of interest, and what you will done after the precipitation step). If really were important, even you could optimize your own working conditions, for your specific working system. One you have identified the main participating variables (e.g., PEG concentration, salt concentration, ph, temp, agitation, etc, etc) you could attempt some type of systematic experimental design to compute a a response surface. This imply to assay multiple variables in at least two different levels rather to test one by one variable. From this surface, you can mathematically derive the optimal working conditions. If you revise the suggestions recevied, you can see there a very ample range de conditions for the same variable. I believe that this cannot be matter of believings or intuitions. Of course, as in any scientific work, it is necessary to spend some time and work to obtain good results.

instead of using buffer, can we re suspend the final pellet with cell maintenance medium (serum free)?