Ammonium sulphate precipitation Problem - Precipitants couldnt be seen on SDS-PAGE and WB (Jan/08/2011 )

Dear all,

i m desperately hoping anyone that could resolve my problem.
i m doing protein expression in pichia pastoris x-33 using pgapz alpha C vector.
upon selection of positive clone, colony pcr was performed and showed the presence of desired band size using pGAP forward and Aox1 reverse primer.

in order to predict the secreted protein, i did a trial experiment using the collected the medium supernatant of 24, 48, 72, 96 hour expression culture and precipitated to 80% saturation. the precipitation was in 1ml s/n culture in a 2ml eppendorf tube and incubated at 4 deg for 2 hr. the mixture was spun down at 10000xg for 30 min. and washed with 90% acetone. then spin again to remove the acetone. then whitish precipitant was air-dried and added with 6x sample buffer and boiled for 15 min. the precipitant was hardly dissolved, bt i still did sdspage analysis, juz to see the presence of protein.
SDS-page shown a single and clear band.

hence, i proceed with 100ml precipitation. and i did a range of precipitation from 20% to 80% saturation.
only 50% to 80% cn produce precipitant.
when i spun down the A.S and medium mixture, there was nth on the bottle wall (juz brownish layer on the bottle wall). bt after 90% ice cold acetone was added, white precipitant started to form in a large amount and increasing for higher saturation.
bz dat was too much of them, so i dissolved them in 1 ml tris-NaCl buffer ph8. and 100 ul of the dissolved solution was added with equal vol. 6x sample buffer. boiling was the same.
bt this time, i gt NOTHING on my SDS-PAGE GEL.

i reli hv no idea why would this happen. i tot the whitish precipitant tat i hv gt is the protein.it is reli a lot. bt i get nth of gel.
or i hv mistaken dat the whitish thg as my protein precipitant?
cn anyone tell me hw protein precipitant look like, and wat mistake i hv done?? i hv nvr seen protein ppt b4.......

i will be very grateful if anyone cn help me out.

Hola, If the AS precipitation runs at low volume, has to work at major scale, but to purify would be better a ion echanger or any specific resin, or if you have enough pure the preparation, you can concentrate by tangential filtration or any ultrafiltration membrane.I dont believe in a salt excess, because you mantain the relation of volumes in the same order than in small scale I suposse, but you can dialyze before put the sample in the PAGE, and the centrifugal force I presume is the same. Other possibility is try precipitate your protein in the isoelectric point or in acidic pH analyzing oellets of pH 5, and 4. Buena suerte

Wait a second... Could you clarify - in your 100mL precipitation - you added 90% acetone to solution that had 80% of saturation of ammonium sulphate or only washed the little bit of precipitate?

i washed a bit of precipitant with 90% acetone

I asked because as good as I remember acetone will produce precipitate when added to ammonium sulphate, it's most probably ammonium sulphate itself precipitating.

General tips - if your protein of interest is not a protease itself, add some protease inhibitors to medium you collected and store at +4 deg.C. You may want to check protein concentration in medium you collected and run whole medium as a control sample at SDS-PAGE.

Regarding your protocol, I find VERY strange to use ammonium sulphate and then wash with acetone. What are you trying to achieve? Is there some acetone-soluble compound you want to remove from the precipitate?

Maybe i hv done it wrongly. but this is wat i read from protein purification book.
the purpose of washing with acetone is to remove the excessive salt.
i hv loaded the whole medium in SDS-PAGE gel, but then nothing could be seen. no desired or degraded bands. i suspect the concentration is too low, thats why i tried to concentrate the medium.

After a quick search I've found only one reference to acetone wash and that is after TCA precipitation, not ammonium sulphate.

As good as I remember from some of my experiments - ammonium sulphate does not work particularly well with low concentration of proteins.

I think you should compare several protein precipitation methods eg. TCA, TCA/acetone, acetone, ethanol or methanol, ammonium sulphate - perhaps one of them would work for you. (Just keep in mind that if you plan to perform some activity assay - some of those methods are not suitable because they may denature your protein.)

For concentrating volumes of 100 mL I would suggest ultratilfration but that can be quite expensive. Other options are lyophilization, or use of dialysis tubing and dry Sephadex (fill tubing with solution and place on dry Sephadex to remove water). If you know pI of your protein you may think about using ion exchange to capture your protein.

thanks so much for ur suggestions. i will try other precipitation methods. bt ultrafiltration wongt be my choice cz reli too expensive.

ultrafiltration membranes and devices can be reused if handled, cleaned and stored properly. if reused then cost becomes more acceptable.

Dear All

I'm working on Aqueous Humor sapmle, in a 2D-PAGE based proteomic project.
actually I tried to precipitate the proteome using Ammonium Sulphate fractionation.I used 20-30-50-70-90 percentages of AS...
I did the procedure according to the common protocols on the web, but unfortunately even after half an hour at 14000 rpm no pellet i found !
also there is a white layer(cream-like layer)floating on the 90%AS aliqout!!

plz someone help me..
should I perform an Acetone prec. prior to AS prec. ?
what can disturb AS prec. ?
thanks in advance.
Farzad