Dear all,
I am using ammonium sulfate precipitation of protein as a first purification step from my crude bacterial lysate.
The method works well, the proteins precipitate in sequential order as controlled by SDS-PAGE, but: The precipitate does not sediment at centrifugation (13k rpm, 15 min), but rather "floats" on top of the solution... I need to soak the "supernatant" beneath this floating pellet... Why is this?
Thanks for any help on this,
Best,
Peter
Dear Peter,
Are you sure that the 'floating pellet' is not just frothing from continuous stirring of the broth??? Also, you might not achieve proper precipitation with the saturation level you are using, I don't know.
Another possibility is that you are adding the ammonium sulfate too fast. Fast addition of the salt causes an uneven distribution of the salt stabilization and leads to denature of proteins. This proteins may be causing your floating pellet.
Hope it is of some help. Best wishes,
Marc
Peter,
I didn't work with bacterial lysates, but I suggest to change concentration of ammonium sulfate. For example, use a 80% saturation, but salt to add fractionally. There is other way. To collect your supernatant and then to add ammonium sulfate in final concentration of 70%.
I suggest you to filtrate the supernatant with the "floating pellet and to verfiy if your protein of interest is included. If the ammonium sulfate is the first step, I suggest you to first use 30% saturation, by adding slowly the salt in the solution, then bring the supernatant to 70% saturation. Then assay the three fractions, namely the 30% pellet, the 70% pellet and the 70% supernatant for your protein of interest.
You have a smart calculator for the ammonium sulfate quantities to use at the following link:
http://www.encorbio.com/protocols/AM-SO4.htm
I've had floating precipitates in higher density salt solutions but it was usually because the composition of the floating material included lipids and the the floating layer was able to pass though a 0.45 micron filter. This layer was not the desired material and could be removed. HIC chromatography may give you more alpha value in your process.
The "supernatant" (which is actually found under the floating precipitate) does not contain the protein of interest, so it is indeed located in the floating matter.
maybe 13k rpm does not suffice to sediment the material?
If it's not air-entrained and centrifugation is required, perhaps because of scale of operation, then higher g force may move the precipitate downward but it could lift up again after the centrifugation is over. Ultracentrifugation may not be practical at large scale. What seems to be the case, if you want to continue with centrifugal methods, is that the density of the ammonium sulfate solution at temperature has created a semi-density gradient centrifugation or isopycnic scenario. In these cases the layers are isolated by puncturing the bottom of the centrifugal vessel and slowly draining the solution where the desired layer reaches the bottom and is collected as a fraction while the other layers are discarded. If this can be done then you might want to then consider further washing of the "floating pellet" or solubilization and re-precipitation but perhaps with a solvent of different diaelectric constant and density as well as a different salt (maybe Hoffmeister) relative to Coulombic interactions in that solvent because there maybe other classes of solutes besides protein present in this " floater".
Hello peter
I had the same problem with amylase from bacteria even if i added ammonium sulphate slowly. Precipitates would form but they would float on the supernatant. It was difficult to collect these precipitates as with little disturbance also they dissolved back in the supernatant......i tried using organic acids and they worked well for me. especially chilled absolute ethanol (previously kept at -20 degrees)
I do not like ammonium-sulphate precipitaion method at all for hydrophobic proteins (please see files; IEF for hydrophobic proteins and Brain-BIN Pig and Purify Brain Lipoamidase). High concentration of sulphate ions seems to reduce the electrostatic interaction and to increase the hydrophobic interaction. Then, the ammonium-sulphate precipitation is not suitable to purification of hydrophobic proteins at all.
Immunological aggregation or binding-reaction seems to be occurred by hydrophobic and electrostatic interactions including glycochain interaction, and all such immunologic methods (RIA, ELISA, and Western blot) become to be not quantitative or determinative at all.
In my experience, ammonium sulphate promotes aggregation of lipid bodies (fat enclosed in a phospholipid monolayer win proteins embedded). These lipid bodies may be natural, like in oleaginous seeds, or animal blood serum and milk serum, but may also be an artefact caused by cell disruptive techniques. If your protein is not in the cream, you should try to remove it with a mechanical fractionation method (flitration, decantation in a separation funnel or some others), if that does not work, you may try adding water saturated butanol to the extract to aid phase separation of the fat.
See this paper as reference, it happens to be in RG but I read it at PMC (PMC4397300). This is really based on very old references when butanol and acetone were routinely used for protein purification, at times were all of the novel and efficient commercial chromatography media was unavailable.
Article A Three Step Approach for the Purification of Alkaline Phosp...
Best wishes,
Rogelio
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