Ammonium Sulfate Precipitation of Proteins Protocol Biochemistry, Molecular Biology, and Cell Biology Protocols >> Ammonium sulfate

precipitation of proteins protocol The following protocol is for secreted proteins. Modify accordingly for other soluble proteins . Spin down cells !. Pour out supernatant into a flas" or bea"er #. Ta"e out a fraction to analy$e on gel %. Add desired amount of ammonium sulfate &ery slowly 'either in a saturated li(uid form, or in powder form) *. Stir for hour

+. Spin down the precipitated protein at ,,,,, - g., #, minutes .. /esupend pellet in PBS for dialysis 'concentrate appro-imately 0+- to 0 ,- based on the amount of the original media) 1. Ta"e some of the supernatant from the spun down protein for dialysis 2esired 3 Saturation , !, #, %, *, ** +, +* ., .* 1, 1* 5, 5* Amount of saturated ammonium sulfate to add 'for ,m4 media) . ml

!.*ml %.!5ml +.+.ml ,ml !.!!ml *ml 1.*.ml !#.##ml #,ml %,ml *+.++ml 5,ml 5,ml

Ammonium Sulfate Precipitation 6&er&iew Going home Ammonium sulfate precipitation is a non7specific purification method that in&ol&es simple purification of proteins from a solution. 8&en though polar and ionic groups of proteins form hydrogen bonds with water in solution, when adding small ions such as ammonium sulfate, water molecules are remo&ed from proteins, allowing protein to precipitate. top Ammonium Sulfate Precipitation Method ' ) Centrifuge ascites fluid at % for #, minutes at #,,,,rpm and transfer the supernatant into a flas". '!) Stir the flas" containing the antibody '% on ice) and slowly add 0# &olume of saturated Ammonium sulfate 'final concentration !*3) and stir for !7% hours. '#) Transfer to a high speed centrifuge tube and centrifuge at % for #, minutes at 1,,,,rpm 'App-. %,,,,9). '%) Stir the flas" containing the antibody '% on ice) and slowly add 0# &olume of saturated Ammonium sulfate 'final concentration *,3) and stir for !7% hours. '*) Transfer to a high speed centrifuge tube and centrifuge at % for #, minutes at 1,,,,rpm 'App-. %,,,,9). '+) Store the supernatant and dry the pellet. '.) 2issol&e pellet by adding :PBS '*,3 of original &olume). '1) Transfer to a dialysis tube and stir for #, minutes at % in :PBS. 'App-. ,, &olumes) '5) /eplace the PBS and stir for hour at %.

' ,) /eplace with !:PBS 'App-. ,, &olumes) and stir o&ernight at % . ';hen conducting 28A8 chromatography purification, use ,mM Tris7<Cl, p<1.* buffer.) ' ) Add thimerosal to ,.,#3 and free$e at 7.,. top Ammonium Sulfate Saturation Method ' ) 2issol&e 1,g ammonium sulfate in ,,ml dd<!6. 'App-. %. M) '!) 2issol&e by heating slightly. '#) Cool at room temperature. '%) Ad=ust to p<.., by adding ><%6< or <!S6%. '*) Store at % following filter sterili$ation.