[ccp4bb] endotoxin calculation help
Dear All, My product is an API that is available in concentration 20mg/ml. We have to determine the endotoxin content of the same. For this I prepared numerous dilutions starting from 1:10 and so on. I found a positive gel clot formation till 1:3000 dilution. Finally in 1:6000 dilution i found a negative result. I take 100 microliter of each dilution and add 100 microliter of lyate. incubate as per specified conditions and then check for clotting. My endotoxin sensitivity is 0.125 EU/ml. from above can anyone help to calculate the endotoxin content in the sample in EU/mg. As per my calculation its between 75 to 125 EU/mg. Is it correct. Please guide me. thanks in advance Megha To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Fwd: bioassay g-csf
-- Forwarded message -- From: megha goyal mgbio...@gmail.com Date: Mon, May 6, 2013 at 1:16 PM Subject: bioassay g-csf To: CCP4BB@jiscmail.ac.uk we perform bioassay of g-csf using m-nfs-60 cell lines but we do not get reproducible results. some time a sample fails and on repeating the assay for same sample it passes. we do not get proper gradation too. can someone please provide me a protocol for g-csf bioassay that has been successfully used. please help me.
[ccp4bb] bioassay g-csf
we perform bioassay of g-csf using m-nfs-60 cell lines but we do not get reproducible results. some time a sample fails and on repeating the assay for same sample it passes. we do not get proper gradation too. can someone please provide me a protocol for g-csf bioassay that has been successfully used. please help me.
[ccp4bb] 10 mM Na Acetate buffer preparation
Our recombinant product is formulated in 10 mM sodium acetate buffer at pH 4.0 and the std composition mentions sodium 0.035 mg and acetate 0.59 mg per ml of sample. It mentions Sodium acetate is formed by titrating glacial acetic acid with sodium hydroxide. Can anyone guide me on how this 10 mM buffer is prepared or on its calculation. What currently I do is the old method that we have been following i.,eUse 0.123 mg Na Acetate trihydrate + 0.476 µl Gl. Acetic acid, adjust pH to 4.0 using 5 N NaOH. But we do not know how these values have been arrived at and if this calculation is correct. Please guide me with this regards. Any help on this will be highly appreciated. thanks, Megha
[ccp4bb] conversion of IU/ml to mcg
We are involved in R D of recombinant filgrastim and the standard sample label mentions it as 30MiOU/ml i.e 300 mcg/ml. How can we determine the IU/ml as we know our protein is 300 mcg/ml. can anyone please guide me on the corelation. regards, megha
[ccp4bb] concentration of protein by TFF
Dear All, we use proflux M12 machine for concentrating our protein using two pellicon 2 casettes PLCC of 0.1 m2. We recently replaced these casettes with new one and now we are facing a problem of low flow rate. i.e earlier when we used to run at a particular pump speed and pressure the flow rate used to be almost double.due to this low flow rate the concentration of protein is taking a long time and results are effected. So kindly guide us what can be the reason for this and what can be done to overcome it. thanking in anticipation megha
[ccp4bb] Solubilization refolding percentage efficiency
Hello, Can anyone let me know how to calculate solubilisation and refolding efficiency. We perform solubilisation and then refolding and check by HPLC if refolding is completed or not [single peak on HPLC]. how do i determine % efficiency of refolding. kindly guide me. regards, megha
[ccp4bb] SP Sep HP tight binding of proteins
Dear All, We used SP sepharose high performance as second stage Ion exchange chromatography for polishing the product. We did get pure product but yield obtained was mere 25%. Our protein has a pI of 5.5 - 6.0 and we had used 25 mM Na Acetate buffer pH 4.5 for loading and same buffer with 1M NaCl for elution, 25 C.V. linear gradient. Can you suggest some changes that i can incorporate to increase the yield i.e additives to be added or some change in pH etc. I tried elution with arginine HCL as elution buffer as was recommended in one paper, but the yield obtained was even less. On washing with 2M NaCl ther is not much peak appearing but on washing with 1M NaOH substantial peak appears. Kindly help me through this. meg
[ccp4bb] Ion exchange protein lost
Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg
[ccp4bb] Sonication e.coli parameters
Hi All, Our protein is expressed as inclusion bodies ibn e.coli W3110 cells. we harvest the cells supend it in water [no buffer] and perform sonication to lyse the cells. we lyse the cells till our O.D is decreased to 1/3rd of original our starting O.D of suspension is about 250. then we centrifuge the abvoe suspencion at 12000 rpm to collect teh inclusion bodies. but we are not getting good yield and also inclusion bodies are not that pure. Do we need to add lysozyme prior to lysis. can anyone suggest a good method to lyse ecoli cells expressing protein in inclusion bodies. Thanks in anticipation meg
[ccp4bb] TCEP effect on protein
Hi all, My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce it (directly added 1 M TCEP to make final volume of 10mM and kept at room temperature for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis buffer is Na acetate pH 4.0]. On performing SDS PAGE analysis after dialysis of the protein we are getting no dimer band, only band of our protein is observed and same is the case with UV reading there is no change in it. But the HPLC analysis of the protein shows two peaks instead of one peak as observed before TCEP treatment. what can be the reason for this. Kindly guide. i need the protein to formulate and conduct stability studies on the sample. the protein we obtain after IEX is pure except the dimer and i do not want to go for SEC as it greatly reduces protein content and also is quite time consuming. any light on what is happening will bevery useful. thanks and regards
Re: [ccp4bb] TCEP effect on protein
Thanks for the all the reply my protein is filgrastim and is biosimilar to Neupogen. it is stable at acidic pH of 4.0. i want to get rid of dimer before i formulate it as we will be maintaining it in liquid form. What i understand TCEP wont work. if i have to use SEC what buffer should i use. My resin is Superdex 75 prep grade and i am using 10 mM Na Acetate buffer containing 150 mM NaCl pH 4.0 for SEC. Once again thanks for all your replies. On 3/26/10, Ho Leung Ng h...@confometrx.com wrote: Does your SDS-PAGE loading buffer contain a reducing agent like beta mercaptoethanol? That could be responsible for the difference between your SDS-PAGE and HPLC results. ho On Fri, Mar 26, 2010 at 5:01 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote: There are 5 messages totaling 490 lines in this issue. Topics of the day: 1. TCEP effect on protein (4) 2. 3 PhD studentships available immediately -- Date:Thu, 25 Mar 2010 22:51:30 -0800 From:megha goyal mgbio...@gmail.com Subject: TCEP effect on protein Hi all, My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce it (directly added 1 M TCEP to make final volume of 10mM and kept at room temperature for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis buffer is Na acetate pH 4.0]. On performing SDS PAGE analysis after dialysis of the protein we are getting no dimer band, only band of our protein is observed and same is the case with UV reading there is no change in it. But the HPLC analysis of the protein shows two peaks instead of one peak as observed before TCEP treatment. what can be the reason for this. Kindly guide. i need the protein to formulate and conduct stability studies on the sample. the protein we obtain after IEX is pure except the dimer and i do not want to go for SEC as it greatly reduces protein content and also is quite time consuming. any light on what is happening will bevery useful. thanks and regards -- Date:Fri, 26 Mar 2010 08:37:43 +0100 From:Ganesh Natrajan natra...@embl.fr Subject: Re: TCEP effect on protein Hi Megha, The two peaks on the HPLC indicate that your protein is existing in a monomer-dimer equilibrium in solution. The dimerisation is most probably caused by disulphide bridges. The use of TCEP is breaking those disulphides and that is causing the equilibrium to move towards the monomeric state. However, when the TCEP is dialysed out, the disulphides start forming again and this is causing the equilibrum to move towards the dimeric state, a process clearly hastened by the strongly oxidising pH 4 of the dialysis buffer. Now it all depends on what you want to do. If you want to use the protein in a (largely) monomeric form, I would recommend that you don't dialyse out the TCEP. regards Ganesh ** Blow, blow, thou winter wind Thou art not so unkind As man's ingratitude; Thy tooth is not so keen, Because thou art not seen, Although thy breath be rude. -William Shakespeare ** On Thu, 25 Mar 2010 22:51:30 -0800, megha goyal wrote: Hi all, My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce it (directly added 1 M TCEP to make final volume of 10mM and kept at room temperature for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis buffer is Na acetate pH 4.0]. On performing SDS PAGE analysis after dialysis of the protein we are getting no dimer band, only band of our protein is observed and same is the case with UV reading there is no change in it. But the HPLC analysis of the protein shows two peaks instead of one peak as observed before TCEP treatment. what can be the reason for this. Kindly guide. i need the protein to formulate and conduct stability studies on the sample. the protein we obtain after IEX is pure except the dimer and i do not want to go for SEC as it greatly reduces protein content and also is quite time consuming. any light on what is happening will bevery useful. thanks and regards -- -- Date:Fri, 26 Mar 2010 08:49:55 +0100 From:Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de Subject: Re: TCEP effect on protein Hi Ganesh and Mega! I do not agree with Ganesh. I assume, Megha, that truly reversed phaseHPLC was used. This is a denaturing method and the natural disulfide should not form again during the run. Also pH 4 can not be described oxidizing. Actually, reduced proteins are often dialyzed against acidic buffers to prevent disulfide formation via the thiolate anion. Still, a reducing agent may be used during the run? Sorry that I can
[ccp4bb] fermentation seed
Dear All We perform fermentation using e.coli clone to obtain recombinant protein. our batch size is 20 L we perform seeeding in two phase i,.e cryovial to seed 1 then to seed 2 and then in fermentation broth. seed 2 is 10% of fermentation broth, and seed1 is 10% of seed 2. incubate each for 10 - 12 hrs. Seed media is LB media and fermentation media is complex media with vitamins I just want to know is there a requirement for two seed, cant we just go from seed 1 to fermentation broth to reduce the passage of bacterial cell. thanks and regards
[ccp4bb] Pulse renaturation
Dear All, we perform refolding of our protein by diluting it 1:10 fold, but we get a low yield in it. I have studied about pulsatile refolding in the book Therapeutic proteins: methods and protocols''. Here it states use of peristatltic pump at flow rate of 0.5 ml / min to add solubilization buffer to refolding buffer and it takes 1 hr to dilute 10 ml sol buffer to 100 ml refolding buffer. but our buffer volumes are large about 1L sol buffer to 10 L refolding buffer. Can we increase the flow rate and what flow rate will be suitable. Also is there any simple spectroscopic method to determine if solubilization is completed or not. since sds page will take a lot of time. thanks and regards.
[ccp4bb] Acidic protein purification
Dear all, our protein is stable in acidic pH at a pI of 6. we do not have any tag in it and it is expressed in inclusion body form. which ion exchange chromatography will suit it cation or anion exchange and do i use a strong or a weak ion excahnge resin. thanks and regards.
[ccp4bb] Solubilization buffer
Hi all, We use 8M urea solubilization buffer for our protein in inclusion bodies and recommended temperature is 10-15º C. but in 8M conc the urea does not dissolve and is in crystalline form only, will it have any effect on solubilzation efficiency. Our solubilization time is 1 Hr and after that we centrifuge and use the supernatant for refolding via dialysis. however the pellet after centrifugation of solubilzation show presence of our protein on sds page analysis. what should we do so that the process of solubilization is complete and our protein is not lost in pellet. thanks in anticipation. meg
Re: [ccp4bb] Inclusion bodies centrifugation
Tried out the method, but our protein is also lost in the supernatant. i collected the supernatant after 1st centrifugation and centrifuged it to obtain the pellet. we observed that our protein is present in that pellet and in further washes also we see protein loss too. Thanks anyways for all the suggestions. On 10/27/09, Artem Evdokimov ar...@xtals.org wrote: IB’s precipitate in a few minutes at max. speed of tabletop Eppendorf. Almost any speed above 5-6K in a larger rotor will precipitate them in 10-20 minutes, provided that the density of your IB wash is normal (i.e. you’re not using 70% sucrose, Renografin, or Cesium Chloride etc). In other terms, no worries. You have the technology. Artem -- *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *megha goyal *Sent:* Tuesday, October 27, 2009 10:21 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Inclusion bodies centrifugation Dear All, Our protein is expressed as inclusion bodies and I want to separate inclusion bodies from E.coli from the cellular debris after* *lysis of the cells by sonication. Can I do this by normal centrifugation? and if yes, at what speed? Our centrifuge has maximum speed of 14000 rpm. Can we do the separation using this centrifuge and if so how. Thanking in anticipation.
[ccp4bb] Inclusion bodies centrifugation
Dear All, Our protein is expressed as inclusion bodies and I want to separate inclusion bodies from E.coli from the cellular debris after* *lysis of the cells by sonication. Can I do this by normal centrifugation? and if yes, at what speed? Our centrifuge has maximum speed of 14000 rpm. Can we do the separation using this centrifuge and if so how. Thanking in anticipation.
Re: [ccp4bb] removal of dimer
Thanks a lot for all your suggestions. But how much DTT or BME shall i add. Also i fear that these will interfere in the formulation and how shall i get rid of them then. As for GPC my protein is 19KDa and diemr will be approx 38 KDa and it is difficult to separate them without losing the protein so i am searching for alternative method. thanks. meg On 2/18/09, Anthony Addlagatta anth...@iict.res.in wrote: Why don't you add some reducing agent like DTT or BME to make everything as monomer? Anthony On Tue, 17 Feb 2009 10:47:28 +, Meg wrote I have purified my protein granulocyte colony stimulating factor using chromatography steps. my protein is relatively pure when analysed by reducing and non-reducing SDS PAGE method. however non-reducing page shows DIMER presence. i have about 250 ml pure protein sample and do not want to perform gel permeation chromatography as it will be time consuming. is there a way to get rid of dimers by some other method. can dialysis help me. kindly guide me as i have to formulate the product and fill it quickly. Thanks in anticipation. meg - Anthony Addlagatta, Ph.D. Ramanujan Fellow and Senior Scientist Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad- 57, INDIA Tel:+91-40-27191583 Url: http://www.iictindia.org/zacb/Dr.%20Anthony.aspx
