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Hi, MBP tag: 1.there might be a TEV cleavage site in your MBP variant. 2. your protein needs salt to stay in solution 3. your protein forms aggregates with MBP GST tag: you probably concentrate a protease together with your protein. You need a protease inhibitor kit to take care of different types of proteases. It looks like a His tag would be a better option for you. Maia vikrant saa wrote: > Dear all > I am working on purification of 14 kd protein(pI 8.3, basic protein) > that has MBP(maltose binding protein, 45 kd,) tag, and same protein in > other vector(pGEX-KT) that has GST tag. During affinity purification > in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer > throughout the purification.I do on column cleavage with TEV to remove > the tag (for crystallization purpose). > Purification with MBP tag: > After cleavage MBP also appear in elution fraction along with cleaved > protein of my interest. To remove MBP(pI 5.0) from protein of my > interest I have to do anion exchange chr. with DEAE resin(weak anion > exchanger) with buffer of pH7.3. Hence I do dialysis against the > buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room. > I have few problems: > 1) why the MBP elute with protein of my interest ( I tried at low salt > concentration also but still elute). > 2) During dialysis my protein of interest precipitate. > 3) If I tried FPLC, protein of interest and MBP elute in same > fraction with superdex 200 column. > > Problem with GST tag purification: it give me impure protein even > after enough washes with high salt concentration buffer (upto 2 > molar). When i concentrate protein in CENTRICON (Millipore, > centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer > with 5mM BME) it degrade very fast and probably aggregate as smear > obtain on SDS page below the size of protein after concentration (even > protease inhibitor not very much helpful) and band intensity of > protein of interest almost remain same before and after concentration > step. > Please send me your valuable suggestion to overcome to these > difficulty. I have also tried with some additives such as sucrose, > glycerol, PBS buffer. > > > With regards > -- > $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ > VIKRANT > Junior Research fellow > Cancer Research Institute > Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), > Kharghar, Navi Mumbai, India > $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ > > ** > ** > > > > > > Send free SMS to your Friends on Mobile from your Yahoo! Messenger. > Download Now! http://messenger.yahoo.com/download.php