Re: [ccp4bb] protein degradation during concentration for crystallization trials
Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to nearly water may not be great thing to do. Can your protein concentrate in your purification buffer to about .1-.2mM? If it can, I highly suggest trying to use the dialysis buttons from Hampton and screen a variety of different pHs, salts and additives to see what your protein can tolerate, before committing an entire purification prep to concentration. I've also noticed if you're using the centricon concentrators, make sure you take it out every few minutes and pipette it the solution a bit. These concentrators tend to create a concentration gradient, making the local concentration of protein very high in bottom of the concentrator, often times resulting in ppt. Hope it helps
[ccp4bb] protein degradation during concentration for crystallization trials
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
Re: [ccp4bb] protein degradation during concentration for crystallization trials
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
