Dear scientists,
I got around 400ug of purified soluble recombinant protein in PBS1x at pH 6.3. I want to remove endotoxin from my protein. I used 2mL of Thermo Scientific Pierce High-capacity Endotoxin removal Resin. I got only 4ug in elution fraction, most of my protein was found in flow-through. Could you give me advice to solve the problem please! Thank you so much.
salut My Dung !
J'espère que tu vas bien! Que deviens-tu depuis tout ce temps ? (8 ans déjà, non ?)
Je ne connais pas la résine Pierce que tu mentionnes, mais je suppose que cette résine est censée retenir les endotoxines, non ta protéine purifiée. Par conséquent je suppose qu'il est normal que celle-ci se retrouve dans le flow through, non ?
Si tu peux me donner des détails sur le protocole...
Bon courage !
Amicalement,
Pierre
Salut Pierre,
Je vais tres bien. Merci pour ta réponse. C'est claire. J'ai mal compris le protocole.
Endotrap blue is what I have used. You pass the protein through the column. It does not stick but it removes all the endotoxins.
You have no problem, the protein should be in the flow-through, only the endotoxins are bound.
You probably purified protein on RP-HPLC column. I recommend for lovering endotoxin level use passing through small amount DEAE sorbent (DEAE-Separose GE) at pH below 0,5-1 units isoelectric point your protein.
Hello,
Endotoxin can be removed with three possible mechanisms:
1. Affinity based : Affi-Prep Polymyxin resin from Bio-rad
http://www.bio-rad.com/en-in/product/affi-prep-polymyxin-resin?ID=5c53a1f3-b0da-4799-8a54-7e94f090e0f9
2. Charge based : MacroPrep High Q ( When your Protein have basic pI and operating at neutral pH) , You can try Ceramic Hydroxy Apatite also.
Selecting right resin for endotoxin removal :
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6813.pdf
3. Size based : If your desired protein size less then 5kda then you can try TFF (10kda) in flowthrough (permeate mode), Endotoxin have higher size so endotoxin will retain in retenate and your product will come in flow-through.
Hope this is useful.
Please contact me for any more info or support.
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