I took a sample from a Mtb culture that is growing in Middlebrook 7h9 enriched with OADC(oleic acid,albumin, dextrose and catalase). The bacteria were harvested by centrifugation, and the supernatant was frozen at -70°c. How can i get rid of all that albumin from the medium. Unfortunately i don't have acess to a synthetic medium like sauton's broth to inoculate the bacteria after MB7h9. Maybe TCA precipitation method? Should i start with a new medium?
My preference would be to use a growth medium without albumin, if possible.
There are resins available for removal of albumin. Of course, there is the possibility that your protein will also stick to the resin. And cost is likely to be an issue.
TCA will precipitate and denature all the proteins in the medium, including the one you want.
You could try ammonium sulfate fractionation. If you are lucky, you may be able to enrich your protein relative to albumin. Then, you could follow up with ion exchange chromatography to further separate your protein from the remaining albumin.
Depending on your protein product stability window ( pI and salt tolerance) you can screen precipitation with pH or Salt.
Depending on pI of your desire protein product , you can choose Ion Exchange chromatography. If pI 3 - 7 go for Anion Exchange with Tris buffer ( pH 7-8) with conductivity < 3ms/cm. If pI is 7-10 then Cation Exchange works well. Buffer (pH 4-6, citrate or Acetate ) with conductivity < 3ms/cm. Elution with linear gradient of 0 to 1M NaCl in same equilibration buffer used.
Hope this helps.
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