what kind of resins is CM-sephadex? what kind of functional group is attached to
ID: 88995 • Letter: W
Question
what kind of resins is CM-sephadex? what kind of functional group is attached to CM-sephadex?
what was the reason to add Buffer C with 0.05 M KCl (LOW SALT) to the resin? and what was the reason to add 0.2 M KCl (high salt ) to the resin? we doing purification by chromatography of Taq protein
We carried out the chromatography in this lab in a conical tube(known as batch chromatography ) instead of in a column (column chromatography) . what are the differences between the two methods? can you see any advantages to either methods?
If u can write at least 5 sentences to each set of question i will love you forever .
Explanation / Answer
CM sephadex is a weak cation exchange resin used in ion exchange chromatography. Weak anion exchangers loose charge when pH increases whereas weak cation exchangers gain charge. Main functional group is carboxymethyl group.Its counter ion is sodium.It works between pH 6 to 10.
The pH and ionic strength of the equilibration buffer are adjusted to favour binding of the molecules of interest in the solute. Weak ion exchangers have their own buffering capacities and need longer equilibration times. At this stage the immobilized groups are associated with exchangeable counter ions.
Next stage involves increasing the ionic strength of the eluting buffer or changing its pH to conditions that favour the elution of the bound substance of interest.
Batch or Pre-Column Chromatography methods are generally fast, easy, have high recovery and have low resolution (purification factor of total protein).Whereas as Column Chromatography Methods require a more extensive process, but generally have higher resolution than Batch Chromatography methods.
Although batch procedures are less efficient than column techniques they may offer advantages in particular cases. When very large sample volumes with low protein concentration have to be processed, the sample application time on a column can be very long and filtration of such a large sample can also be rather difficult to perform. Binding the sample in batch mode will be much quicker and there will be no need to remove particulate matter. A batch procedure can also be an attractive approach if high sample viscosity generates high back-pressure in a column procedure or if high back-pressure is generated by contaminants such as lipids, which may cause severe fouling and clogging of the column.
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