Hi, can anyone guide me with this query of; In order to obtain apo-form of α-LA, is by adding 3-5 mM EGTA to the solution of holo-α-LA (Ca2+bound). This solution was dialysed against the several changes of 0.1 M KCl solution at pH 7.0 and 4 °C”
1.How can we check the real content of calcium in the protein samples? 2. Do 0.1 M KCl solution contains enough calcium to saturate α-LA during the dialysis procedure. 3. At pH 4.5 (ie, the isoelectric pI of α-LA) part of the calcium will be displaced by protons but exact calcium content in the α-LA sample is unknown.
One way is to submit the sample to a contract lab for ICP-MS analysis of metal content.
Another way is to measure the Ca2+ content using an ion selective electrode, or a fluorometric calcium dye, depending on the type of equipment you have available.
Hi Adam B Shapiro Thanks, umm but even after using EGTA at such high conc. amount we can expect not to have ca2+ removed completely?! like not experimental way but analytical. is there also any role of 0.1M KCl buffer (my second part of question).
EGTA binds Ca2+ very tightly. If the Ca2+ is in equilibrium with the solution to any degree, it will eventually be chelated and will be effectively unavailable for binding to anything else.
As for KCl, it depends on how pure it is. Some of the more expensive grades of common reagents supply an elemental analysis. (example: https://www.sigmaaldrich.com/catalog/product/mm/104936?lang=en®ion=US)
You can also remove any minor contaminating divalent metals by passing the solution through Chelex resin.
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