With acetic acid serial dilution i take an effort to detect the isoelectric point of an enzyme. I observed precipitation in PH 2.85 and 3.1.Of them which one want to taken as isoelectric point?plz help me
no idea ,i also try protein isolation and identification by hplc
In order to determine isoelectric point (PI), you need to get the pKa form the acid-base titration curve if you get 2 pkas then simply, the isoelectronic point will be the average of, these two pKas, i.e. pI = 1/2 (pKa1 + pKa2).
The standard method to measure the isoelectric point of a protein is isoelectric focusing.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/isoelectric-focusing.html
You can also predict the isoelectric point of a protein if you know its sequence:
http://scholarworks.rit.edu/cgi/viewcontent.cgi?article=9081&context=theses
I would also encourage you to see and try other software available out there besides the above method. See if their results agree.
Unfortunately i dont have the sequence and i have to do it manually. Glacial acetic acid serial dilution is working. But i observed precipitation in two tubes which have low ph. Plz anybody help me to solve this problem
Precipitation isn't a particularly good indicator. I'd cut out the hassle and use an IEF gel. (eg. ThermoFisher... they supply a kit with standards).
Precipitation may occur because of protein denaturation at the acidic pH, rather than because the protein is at its pI.
A protein may not precipitate at its pI. It has zero net charge, but still has positive and negative charges, as well as uncharged polar side chains, that can keep it in solution.
Computer programs that predict pI based on sequence can be inaccurate because they don't take into account the environment of the charged residues, which can have large effects on their pKas.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry