Dear friends, When I try to measure protein encapsulation efficiency & release estimation from CSNP using mBCA, I get very high back ground due to chitosan residues as they have free NH2 group, which also react with mBCA reagents.This mask the amount of protein present in the supernatant and make it almost impossible to estimate protein. No luck even with 10000X dilution of supernatant or even filtering the supernatant with 0.45 micron filter or even 3kD cutoff filter. Has any one ever experience the same problem? Chitosan community, please do suggest some solution what to do in this case. I will greatly appreciate your kind help and guidance in this regard.
Have you tried other methods of protein estimations?
if you have adequate amounts of protein, methods that do not rely on -NH2 groups may be much more useful. For example, you are not likely to have too high an absorption at 280nm or perhaps even for the peptide bond at 218nm. Success of quantitation of protein will depend on how well defined is your system so you can fully and accurately account for absorption by other components.
Dear Tausif Alam, thank you so much for your technical insight into this very problem Previously I tried UV absorption at 280 and 215 as well but without luck as my protein amount after release seems to be very less. I will definitely try to increase the amount of protein encapsulated nano particles so as to get sufficient protein for UV absorption assay. I nay have to modify the estimation protocol as Acetic acid tends to cause continuous fluctuation in the absorbance even at low concentration of less than 1%.
Another thing I noticed in the literature that people have used BCA method for the estimation of protein encapsulation efficiency as well as release profile from chitosan nano and micro particles, which is kind of beyond my understanding, as in my case chitosan even at 10000X dilution as well as filtration gave so much of high background that any non-encapsulated protein in the supernatant as well as released protein is going to be masked and definitely be overestimated by a magnitude. The amount of background from chitosan residue will vary depending upon the acetylation level to big extent with a positive correlation between DDA and high back ground due to more number of available CONH groups on chitosan polymer but I am not sure what is going on in this case, just wondering, without jumping to any conclusion.
You can also try with other encapsulation methods and then compare the release with your 10000X dilution for the same, then u can come to conclusion i guess
Yes, I used to have the very same problem with you, and we even have exactly the same idea with the filtration thing, but unfortunately we can not execute the filtration approach due to the financial & time limitation of our project. I have one more option that I haven't tried though, that is to tag your protein with Fluorescent molecule (e.g. FITC), then measure the protein:FITC absorbance. So you can measure the amount of protein by measuring the FITC trapped along with protein inside the nanoparticle.
Hope that help,
Radif
Dear S. Thanigaivel, Thanks for your kind suggestion. But as I mentioned, the real problem is that there is no difference in the Blank and the encapsulated supernatant even after 10000X dilution. So you can hardly conclude any thing from this, or even I guess will not be possible to even compare it with another encapsulation systems.
Dear Radif, good to know that I am not alone who is facing this kind of problem. Thank you so much for your kind suggestion. This is what I am planning to do to see if we can get the protein encapsulation efficiency as well as release estimation under our system. Thanks to both of you once again.
Possibly you can try it with a proteingel, stained with coomassie or other method. With that, you get additional information about size (degradation) and when you let your protein run in several defined dilutions, you can measure the density, prepare a standard curve and calculate the protein amount.
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