Hi,
I am working on Ni-NTA gravity column chromatography to purify my protein, I am confused with the different between column volume and bed volume. What is the different between those two?
Thanks
Dear Sir. Concerning your issue about the different between column volume and bed volume in Ni-NTA gravity column . The Ni-NTA Resin is specifically designed for the purification of recombinant proteins fused to the 6 x histidine (6XHis) tag expressed in bacteria, insects, and mammalian cells. The resin is high affinity and selectivity for recombinant fusion proteins that are tagged with six tandem histidine residues. The Ni-NTA resin can be used to purify 6X His tagged proteins under native and denaturing conditions. Proteins bound to the resin can be eluted with low pH buffer or competition with imidazole or histidine. The Ni-NTA resin uses nitrilotriacetic acid (NTA), a tetradenate chelating ligand, in a highly cross-linked 6% agarose matrix. The NTA binds Ni2+ ions by four coordination sites. The total volume of the columns is indicated in the table above. For sample volumes larger than the column capacity, simply perform multiple applications and centrifugations until the entire sample has been applied. Ensure that the capacity of the column is not exceeded. The total volume is equivalent to two resin bed volumes (RBV). For moredetails, I think the following below attached file may help you in your issue.
Thanks
One and the same thing. It refers to the volume of resin packed which can be calculated by taking height of the resin (and not the tube) and putting it in the formula
v= π r2 h
r is inner radius of column
h height of the resin packed.
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