Chemical Forums
Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: giustina53 on October 07, 2012, 05:32:21 PM
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Hi! In the lab, we did an ion exchange purification which is purification based on charge. My teacher asked this question:
If you were purifying acid phophatase by this method, how will you know which fraction/s contained this enzyme?
I know that you use a concentration gradient and that the weaker charged proteins come off first because they get replaced by the lower diluted salt quicker and that proteins with more charge get eluted at higher salt concentration because since they have more charge they bind to the column more strongly and aren't replaced by the salt until a higher concentration.
So can I figure out when the acid phospahatse will be eluted into which fraction just based off how many charges it has (how strongly charged it is) and, therefore, which fraction it will be in?
Only problem is that I can't find the charge on acid phosphatase.
Help?! ???
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Although you have the right idea for how to predict where the enzyme will elute, in general, it can be difficult to predict exactly when your protein will elute from a column. Therefore, biochemists will generally test the various fractions for enzymatic activity to determine which fractions contain the enzyme.
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It is not uncommon to screen all fractions by some method that detects all proteins, such as the Bradford assay or A(280), then to follow this up with enzyme assays. When an enzyme assay is not possible, one sometimes uses SDS PAGE on individual fractions.
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We used A(280) to screen all the fractions. Does anyone know the A(280) of acid phosphatase or where I can find it? I'm assuming the literature but it takes so long to go through all the research papers. Maybe someone knows off hand? :P
Thanks for the help guys!
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If you have the amino acid sequence of your protein, you can estimate the extinction coefficient using various online tools, for example at http://web.expasy.org/protparam/
Note that A280 does not distinguish between different types of proteins, so if you have two different peaks coming off the column, you could use the A280 measurements to say that there were two peaks, but you could not determine which of the two peaks contained your protein. If you know the molecular weight, running SDS PAGE on the fractions as Babcock_Hall suggested is a good idea as you can use the size of the proteins to identify which of the two peaks contains your phosphatase.