I conjugate one protein and one peptide to the polymer iron oxide coated nanoparticle and I am going to determine the protein concentration in my solution, but I do not know how to determine their conjugation efficiency and their concentration in the solution. Does chromatography work here and if it works then which type of chromatography has application here.
Rozita, you will need to separate the two proteins first (bound and not bound). Use gel filtration if there is a different in size larger than 500 Da. There are different resins you could use to separate by size. Collect the fractions separately. Your unbound protein should come out of the column first because it should be smaller. The bound protein will come later because its weight should be larger. Pool the fractions (depending on how much volume, they will be spread out), then salt- precipitate the unbound protein (Magnesium Sulfate works for this purpose). Now that they are separated, you should be able to measure protein concentration using a protein assay. Just check that the iron oxide does not interfere and you are all set.
Why do you want to go for chromatography? If you are looking at a simple mixture as described, I would rather look toward the regular set of UV-based protein assays (Bradford, BCA). You will need to check for dynamic range and buffer compatibility, of course. For determination of the peptide concentration HPLC-UV might work nicely, again provided your concentrations are sufficient for the detector and you have a quantified standard in hand (which I suspect you have for the experiment you are doing).
Any colorimetric assay either Bradford or BCA works to obtain direct quantification, make sure to conjugate your BSA standard with nano particles as well. I personally liked Bradford because it was not influenced by the presence of iron oxide. BCA was slight affected by the presence of iron in suspension, although the effect was quiet linear. So you can try both. You can also use TR-LRET to measure protein aggregation. I'd like to see high-affinity MRI probes for accurate and precise measurement! If you have access go for it! G'Luck!
I would determine the protein concentration by UV, before the conjugation.
After the conjugation I would dialyse your system with a suitable MW cutoff in order to remove the excess of peptide.
A simple gel SDS-PAGE can maybe be enough to understand the proportion of conjugated/non-conjugated protein
I have two different protein nature stuff in my solution and I am confused that whether I can determine their concentration using Bradford assay or not? Because Bradford assay is Colorimetry assay and the color will change based on the presence of protein in solution and now I do not know how I can be sure that both of proteins conjugate effectively and how I can determine concentration for each of them. Based on my understanding, Bradford assay only will show total concentration in my solution and it is not able to give me the exact concentration for each proteins in my solution. If I am wrong please give me correction. very appreciative
You need to do the conc. assay (UV Bradford whatever) before mixing up your proteins. It does not make sense to assess the conjugation yield simply by measuring the final concentration. Am I right? You need to find a way to see A, B, A+B together. I Think of an SDS page gel. If the conjugation is covalent the system will run higher than the two.
Dear Codutti
Your right and SDS page is the best for detection of A and B because it will run in higher place, but I am looking for the method to determine A and B concentrations in A+B solution after conjugation. Do you think that chromatography will work here? If YES then which type of chromatography has application here to find out A and B concentration after conjugation? If you know another useful method please share it with me. Thanks
Rozita, you will need to separate the two proteins first (bound and not bound). Use gel filtration if there is a different in size larger than 500 Da. There are different resins you could use to separate by size. Collect the fractions separately. Your unbound protein should come out of the column first because it should be smaller. The bound protein will come later because its weight should be larger. Pool the fractions (depending on how much volume, they will be spread out), then salt- precipitate the unbound protein (Magnesium Sulfate works for this purpose). Now that they are separated, you should be able to measure protein concentration using a protein assay. Just check that the iron oxide does not interfere and you are all set.
Nice answers above, but why not make it easy? Go for IR. I am using the Direct Detect from Millipore. It works nice and you can see the IR spectrum in addition, therefore, you can make conclusion on the amount of bound and non-bound species.
best
Nice reply but it is not entirely precise. In size exclusion chromatography bigger particles exit the column before small particle due to the fact that larger particles are excluded from the matrix and are eluted faster than small particles that can enter the gel pores more deeply and are eluted slower.
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