[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

[ccp4bb] Fwd: bioassay g-csf

-- Forwarded message -- From: megha goyal 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

[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 us

[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 b

[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

[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

[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 i

[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 centrifug

Re: [ccp4bb] TCEP effect on protein

> > > 1. TCEP effect on protein (4) > > 2. 3 PhD studentships available immediately > > > > ------ > > > > Date:Thu, 25 Mar 2010 22:51:30 -0800 > > From:megha goyal > > Subject: TCEP

[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 a

[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 -

[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 bu

[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 w

Re: [ccp4bb] Inclusion bodies centrifugation

t; 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 &

[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 d

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