Hey, i am trying to see the effect of various matrices on encapsulation of my enzyme. For a start, i am doing it with BSA and i tried the method described by Mohidem and Mat (2012). The problem is when i finish sonicating TEOS with water and HCL i see 2 phases, one clear at the bottom and white gel like at the top. I am not sure if this is normal, after that when i add my protein with the buffer, it doesnt gell in 5 minutes (as described by the paper). I am not sure where iam going wrong, what i do see is that after addition of the buffer the white layer is on the top and the clear at the bottom. and the gel doesnt form its still runny in consistency. Please help.
Usually on the sol-gel process is added water and alcohol for the phase transition. On the work mentioned by you the alcohol is missing, I suppose they used the alcohol resulted from the hydrolysis for phase transition.
If you do not want to put some alcohol, you have to stir your sol until the opacity of the solution disappear and the phases will not separating after you stopped the stirring.
If you put some minor amount of alcohol on your sol the phase transition will occur more easier and after approximately one hour you could sonicate your sample and the gelation will occur in a few minutes
Thank you zoltan, yes they do not use alcohol because it seems that if you sonicate this mixture you dont need to add alcohol. Maybe i should try sonicating at a higher frequency. So you are saying i shouldnt obtain two layers after sonication? Probably the teos remains at the bottom?
As I see the key of your success is the obtaining of the transparent solution without phase separation. Until you do not obtain the mentioned solution, when you will sonicate (your solution) the phase transition will occur. At first I would try to stir for longer period the solution before the sonication, That longer period could 2-3 hours but could be 24 if it is needed.
Thank you will, try doing this. So in the end the solution must be transparent not opaque right?
Yes that's right, until the solution is opaque, the phase separation will occurs.
Hey zoltan, can you check these images? Is this how the gel looks after it forms?
The gels have to be uniform, you had on the pictures phase separation. In this way I think is not proper for enzyme immobilization, It is a risk that the enzyme or a part of the enzyme will remain in the upper part and as I see the gel is opaque.
Why do you insist with this enzyme immobilization protocol? Why is it so important to not have alcohol. I think if you want alcohol free gels, you could have it in the following way: Put 1 mL of ethyl alcohol at the start of the synthesis using the same condition, after an hour of stirring heat up until 80 degrees your sol and the alcohol will evaporate, than you could sonicate your sample
Hello Zoltan, there is a very important reason for not using an alcohol, as you know it denatures protein. Also, the publication method i am following they havent use it which is why they are sonicating the sol. But they havent mentioned what strength they used because when i tried it at level 5 for an hour instead of half hour i obtained a clear solution however i could still see a partition in the end.
I think as I said before the key in your case not the sonication energy (the length of the sonication, the amplitude or the pulse on/off time), the key is the homogeneity of your sol, try to stir longer time like 24 or 48 hour if in 5 or 8 hours is not enough.
I am glad , i have someone to guide me! I am so new to this silica gel and stuff. I will try this thanks zoltan
yes zoltan! finally i obtained what i wanted, i stirred it for a period of 4 hours and obtained a clear elastic solution
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