Technical Note: TNCFGLYNXAMSULF 1012

Large Volume Precipitation of Proteins with Using Thermo Scientific Fiberlite Carbon Fiber Rotors

Key Words Ammonium Sulfate Precipitation, Superspeed Centrifuges, Carbon Fiber Rotors

Introduction Ammonium sulfate precipitation is a method of by altering the of protein. Ammonium sulfate is commonly used due to its high solubility that allows solutions with high ionic strength. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution. Differential precipitation of proteins by ammonium sulfate is one of the most widely used preliminary purification procedures. It is based on proteins having differing solubility in ammonium sulfate solutions and can result Figure 1: Thermo Scientific Fiberlite F12-6x500 LEX in a two- to five-fold increase in specific activity. carbon fiber rotor. Provided that appropriately buffered ammonium sulfate solutions are used to protect the desired activity, 2. While gently stirring, slowly add ammonium sulfate to recoveries approaching 100% can be expected. A typical 45% saturation (277 g ammonium sulfate/liter solution). protocol consists of adding ammonium sulfate to give Stir gently for a minumum of 4 hr and a maximum of specific percentage saturation, followed by a period of 16 hr at 4 ºC. time for proteins to precipitate and a centrifugation step to collect the precipitate. The adapted protocol1 Note: Addition of ammonium sulfate acidifies the solution, described below was carefully followed to precipitate therefore protein stability may be improved by buffering protein removed by centrifugation using the Thermo with at least 50 mM HEPES or Tris buffer. It is important Scientific Sorvall LYNX 6000 superspeed centrifuge and that no foaming of the solution occurs during ammonium the large volume Thermo Scientific Fiberlite F12-6x500 sulfate addition as this will cause denaturation of proteins. LEX carbon fiber rotor. The total capacity of the If foaming occurs, slow the addition of ammonium sulfate. Fiberlite® F12-6x500 LEX rotor is 3 liters and is rated Make sure that the ammonium sulfate dissolves nearly as for RCF up to 24,471 x g. quickly as it is added. The ammonium sulfate concentration was increased 3. Transfer the ammonium sulfate precipitated solution to a value that will precipitate most of the protein of to 500 mL Thermo Scientific Nalgene polycarbonate interest, while leaving the maximum amount of protein centrifuge bottles with screw caps (PN 3140-0500). contaminants still in solution. The precipitated protein of interest was recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.

PROTOCOL: Ammonium Sulfate Precipitation

1. Measure the volume of protein solution to obtain 1500 mL. If necessary add 1 M HEPES, pH 7.4, or 1 M Tris-HCl, pH 7.5, to a final concentration of 50 mM. Perform centrifugation using theSPANISH Fiberlite Conclusion thermoscientific.comF12-6x500 LEX rotor and the Sovall® LYNX 6000 This protocol is useful to quickly remove large amounts superspeed centrifuge with the following parameters: © 2012 Thermo Fisher Scientific Inc. Reservados todos los derechos. Las otras marcas comerciales son propiedadof contaminant de Thermo Fisher Scientific proteins, Inc. y sus as done in the first step in many filiales.25,000 Características, x g condicionesfor 20 miny precios at están 4 ºC.sujetos a posibles cambios. Algunos productos no están disponiblespurification en todos los schemes. países. Consulte It los is also often employed during the detalles con su representante comercial local. later stages of purification to concentrate protein from a 4. Decant the supernatant, being sure not to disturb the 2 Alemania +49 6184 90 6000 Francia +33 2 2803 2180 dilute solutionRusia +7 812 following 703 42 15 procedures such as gel filtration. Australiarelatively +61 39757 loose 4300 protein-ammoniumHolanda sulfate +31 76 pellet. 579 55 55 Suiza +41 44 454 12 22 Austria +43 1 801 40 0 India +91 22 6716 2200 In additionUK/Irlanda to the +44 rapid870 609 precipitation 9203 of protein Note: At this point, the pellet can be stored for several Bélgica +32 53 73 42 41 Italia +39 02 95059 554 contaminants,USA/Canadá the +1 Fiberlite866 984 3766 F12-6x500 LEX rotor will Chinaweeks +86 at 21 4 6865 ºC 4588without or significant lossJapón of+81 protein 45 453 9220 stability. +86 10 8419 3588 Nueva Zelanda +64 9 980 6700 harvestOtros bacteria países deand Asia yeast +852 2885from 4613 cultures in shorter run The supernatant can also be stored at 4 ºC for later España/Portugal +34 93 223 09 18 Paises Nórdicos/Bálticos/CIS times thanOtros paisesrotors +49 with 6184 similar 90 6000 total volumes. analysis. +358 9 329 10200 o +33 2 2803 2180 References 5. Re-suspend the pellet in PBS, pH 7.2. The solution will 1. Elder, G.H., Tovey, J. A. and D. A. Sheppard, (1983). remain turbid, but discreet flocculent is not visible. Purification of uroporphyrinogen decarboxylase from 6.thermoscientific.com Remove residual precipitate and collectITALIAN the clarified human erythrocytes. Immunochemical evidence for a © 2012proteins Thermo Fisher by performingScientific Inc. Tutti i centrifugationdiritti riservati. Tutti gli altri with marchi thesono difollowing proprietà di Thermo Fishersingle Scientific proteinInc. e delle suewith filiali. decarboxylase Specifiche, activity in human condizioniparameters: e tariffe possono 25,000 subire variazioni. x g forNon tutti 10 i prodotti min sonoat 4disponibili ºC. in tutti i paesi. Per maggiori dettaglierythrocytes consultare il rivenditore and locale liver. Biochem. J. 215: 45-55. 7.Australia Remove +61 39757the clarified 4300 proteins to Indiafresh +91 tubes 22 6716 and 2200 store at 2. Koch,Svizzera U., Choksi, +41 44 454 S., 12 Marcucci, 22 L. and R. Korngold, Austria +43 1 801 40 0 Italia +39 02 95059 554 UK/Irlanda +44 870 609 9203 Belgio4 ºC +32 for 53 up73 42 to 41 one week. Olanda +31 76 579 55 55 (1998).USA/Canada A Synthetic +1 866 CD4-CDR3984 3766 Peptide Analog Cina +86 21 6865 4588 or Nuova Zelanda +64 9 980 6700 Enhances Skin Allograft Survival Across a MHC Class II 8.+86 Alternatively, 10 8419 3588 if column purificationEuropa is delgoing Nord/CIS to be Altre Nazioni Asiatiche +852 2885 4613 Barrier. Journal of Immunology, 161: 421-429. Franciaperformed +33 2 2803 immediately, 2180 dialyze protein+358 9 329 against 10200 column Nazioni non in elenco +49 6184 90 6000 Germania +49 6184 90 6000 Russia +7 812 703 42 15 o +33 2 2803 2180 Giapponebinding +81 buffer. 45 453 9220 Pass the solution throughSpagna/Portogallo a 0.45 +34 μm 93 filter 223 09 18 and apply to the appropriate column.

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Australien +61 39757 4300 Indien +91 22 6716 2200 Russland +7 812 703 42 15 Belgien +32 53 73 42 41 Italien +39 02 95059 554 Spanien/Portugal +34 93 223 09 18 China +86 21 6865 4588 or Japan +81 45 453 9220 Schweiz +41 44 454 12 22 +86 10 8419 3588 Niederlande +31 76 579 55 55 UK/Irland +44 870 609 9203 Deutschland Nationale kostenlose Hotline Neuseeland +64 9 980 6700 USA/Kanada +1 866 984 3766 0800 1 536 376 Nordeuropa/Baltikum/CIS Deutschland International +358 9 329 10200 Andere asiatische Länder +852 2885 4613 +49 6184 90 6000 Österreich +43 1 801 40 0 Andere Länder +49 6184 90 6000 Frankreich +33 2 2803 2180 oder +33 2 2803 2180

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Allemagne +49 6184 90 6000 France +33 2 2803 2180 Russie +7 812 703 42 15 Australie +61 39757 4300 Inde +91 22 6716 2200 Suisse +41 44 454 12 22 Autriche +43 1 801 40 0 Italie +39 02 95059 554 UK/Irlande +44 870 609 9203 Belgique +32 53 73 42 41 Japon +81 45 453 9220 USA/Canada +1 866 984 3766 Chine +86 21 6865 4588 or Nouvelle-Zélande +64 9 980 6700 +86 10 8419 3588 Pays Bas +31 76 579 55 55 Autres pays d’Asie +852 2885 4613 Espagne/Portugal +34 93 223 09 18 Pays nordiques/baltes/CEI Autres Pays +49 6184 90 6000 +358 9 329 10200 ou +33 2 2803 2180

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Australia +61 39757 4300 India toll free 1800 22 8374 Russia +7 812 703 42 15 Austria +43 1 801 40 0 India +91 22 6716 2200 Spain/Portugal +34 93 223 09 18 Belgium +32 53 73 42 41 Italy +39 02 95059 554 Switzerland +41 44 454 12 22 China +86 21 6865 4588 or Japan +81 45 453 9220 UK/Ireland +44 870 609 9203 +86 10 8419 3588 Netherlands +31 76 579 55 55 USA/Canada +1 866 984 3766 France +33 2 2803 2180 New Zealand +64 9 980 6700 Germany national toll free 0800 1 536 376 Nordic/Baltic/CIS countries Other Asian countries +852 2885 4613 Germany international +49 6184 90 6000 +358 9 329 10200 Countries not listed +49 6184 90 6000

TNCFGLYNXAMSULF 1012