Re: [ccp4bb] protein aggregation
Dear Gauri This may be the saturation point of your protein and beyond this it will start precipitating in a particular buffer. Try to set the crystallization trial (if it is your application ) and select those condition in which you see clear drop. Change the purification buffer with that condition buffer. It may work .. Else you can use Sucrose (0.5-2M), PEG6000 (5-10%), glycerol (5-10%) etc in different combination. all d best Vikrant --- Senior Research Fellow (CSIR) Varma Lab Cancer Research Institute Advance Centre for Treatment, Research and Eduction in Cancer (ACTREC) Tata Memorial Hospital, Kharghar, Navi Mumbai-410210 From: gauri misra kamga...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 23 March, 2011 11:11:55 PM Subject: [ccp4bb] protein aggregation Hi, What are the different methods to prevent protein aggregation while concentrating so as to increase the concentration of the protein? I have some idea of adding EDTA and charged amino acids like L-Arg and L-Glu. I would appreciate if the readers share their experiences. Thanks! Gauri
Re: [ccp4bb] Glutathione sepharose
use the following procedure. 1) immediately after use pass freshly prepared cold reduce glutation 20mM , 1mM EDTA in 50mM tris ph8.0. 2-3 CV, 2) wash alternate with buffer 1 and buffer 2 (GE lifescience), 2 cv, 4-5 times, then 1M Nacl 2-3 cv 3) wash with pbs , 5 CV 4) wash with 1% triton, 3 CV 5) wash with PBS, 5CV 6) wash with 70 % alcohol 5 Cv 7) wash with DW 10 CV 8) store in 20% ethanol. dont use GuHCL or urea unless absolutely require . Try to regenerate the beads on the same day of use. Vikrant Junior Research Fellow (CSIR) Lab No. 101, Dr. Varma Lab Cancer Research Institute Advance Centre for Treatment, Research and Eduction in Cancer (ACTREC) Tata Memorial Hospital, Kharghar, Navi Mumbai-410210 # From: Mirek Cygler mi...@bri.nrc.ca To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 2 November, 2010 9:16:37 PM Subject: [ccp4bb] Glutathione sepharose Hello, For various reasons we are frequently expressing proteins with a GST tag. The glutathione sepharose beads that we are using for affinity purification seem to be difficult to regenerate and we see much lower capacity when used the second time. We are following the manufacturer's instructions for regeneration but this not very effective process as opposed to NiNTA which can regenerate multiple times. Due to this, the purification of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that can be successfully regenerated several times? Any comments will be greatly appreciated. Mirek
[ccp4bb] Regarding quality of protein for crystallization
Dear all Thanks for yours valuable suggestion. Just some addition information to make the query clear. 1)My protein has 14 cysteine residues. 2)It is not a metal binding protein. 3)I have added the protease inhibitor cocktail + PMSF during sonication and cleavage. I saw only one band of protein bind on beads. Two band appears after cleavage. 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in Rosetta 2DE3 5) I have to do MALDI and MS MS to know exact molecular weight and sequence that differ in both the proteins. Most of the peaks in peptide mass fingerprint match exactly while some are at different position and size. Amino acids sequence similarity in both the cases are upto 1-405 amino acids (as per limitation of peptide mass fingerprint it show some region upto 1-405 matching in both the proteins). I have certain doubt/solution(s) on the basis of yours feedback and above information. 1) How come PTM can play role when protein expressed in bacteria. 2) TEV is a very specific protease hence non specific cleavage less likely occur. 3) Cysteine residues or temperature dependent expression may be one of the reason of two band of protein. Further suggestion to get rid of this problem will be highly appreciated. With Regards Vikrant
[ccp4bb] Regarding quality of protein for crystallization
I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX vector.I do the expression (induction 24 degree 16 hrs) and purification from Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity chromatography purified protein (cleaved protein after removal of tag) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern being retained after FPLC. Intensity of both the bands remain same even after one or two week of storage at 4 degree. Peptide mass fingerprinting (after trypsin digestion) suggest both are my proteins except minor difference in some peptide peaks. Is it because of rare codon/degradation of protein of interest or any other possibilities? Can I use this mixure for crystallization? With Regards Vikrant
Re: [ccp4bb] protein turns brown
sometime it does happen becoz of protein aggregation, or reducing environment. but it may be your protein color as well that visible during concentration Vikrant From: sandeep toskgu...@rediffmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 24 September, 2010 2:04:54 PM Subject: [ccp4bb] protein turns brown Dear all, I have purified protein from E.coli. expression system. the protein has been purified with three independant columns. Now during concentration step using amicon, the protein shows brown colour. what could be the reason. best regards and Thanks, sandy
[ccp4bb] software for predicting protein solubility, stabililty and disorders
I want to do cloning of a 40 Kd protein in pRSETA, and pGEX-KT vector. I don't have any idea about protein solubility, its multimeric form, stability and disorder etc. There is nothing known in the literature also. Is there any software that can predict these parameters, so that i can decide which domain i need to clone for soluble and stable protein purification. Vikrant Junior Research Fellow (CSIR) Lab No. 101, Dr. Varma Lab Cancer Research Institute Advance Centre for Treatment, Research and Eduction in Cancer (ACTREC) Tata Memorial Hospital, Kharghar, Navi Mumbai-410210 #
[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
