I will be much thankful to anyone if can kindly answer my question,
I'm purifying a metal binding protein from the blood plasma of a sea squirt. The method is briefly as follows: Blood plasma is precipitated by 80 % saturation of ammonium sulfate and then purified by DEAE ion exchange and Sephacryl gel filtration chromatography. Ammonium sulfate precipitate and active fractions in chromatographic steps are lyophilized. My question is how do I calculate the yield of each step since initial blood plasma is in a liquid state.
Looking forward to a kind reply...
Hi Minoli, the heart of every purification procedure and in biological research in general is having an assay that you trust.
I assume that you have a trusted assay. The yield is easy to calculate from the protein contents of the solutions generated. Ignore the liquid state of your solution. Report your results in terms of mg/ml of protein.
I agree with Steingrimur Stefannson about the importance of a trustworthy, quantitative assay. Yield in a purification table is usually calculated based on the total number of units of activity at each step. The sample in the first step is always a liquid of some kind. Frequently it is a homogenate of a solid, but it doesn't matter. Don't worry about that. Just make sure you measure the volume of the sample at each step, which you will need to know in order to calculate the yield.
You will need to define a unit of activity. If possible, let the unit definition be the standard 1 micromole of product formed per minute. For example, suppose you find that 10 µl of a 1:1000 dilution of sea squirt blood plasma produces 1 µmole/min of product in your assay. The total activity is therefore 1 µmole/min/0.01 ml x 1000 = 100,000 units/ml. If you started the purification with 2 ml of plasma, then the original sample had 200,000 units of activity. This is the denominator for the yield calculation. At the end of the purification, if you have 4000 units left, the yield is 2%.
I would presume that you would add the metal to your plasma beforehand and let bind with your protein (like Fe in Hemoglobin). Then at each purification step trace and determine the concentration of the metal either by MS or scintillation counter (if an isotope).
If you have an antibody specific to your protein, then you could set up a quantitative immunological assay, an ELISA, that would allow you to quantify the yield of the protein through the purification, even without a purified protein standard. This could be used if the protein does not have enzymatic activity. If you do not have an antibody but know the sequence of the protein, you can have an antiserum prepared commercially against a peptide corresponding to an antigenic part of the sequence.
A metal binding assay, as mentioned by Bruce Neagle, is also a possibility. Another way to do it is by a competitive binding assay with a fluorescent probe for that metal. Here is a catalog chapter on such probes:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-indicators-for-zn2-and-other-metal-ions.html
Thank you very much, dear sir, Steingrimur Stefansson, Adam B Shapiro, Bruce Neagle and Adam B Shapiro for your valuable answers. It is a great help.
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