I'm trying to immobilize cellulase on Fe3O4 magnetic nanoparticles coated with Tannic acid, with glutaraldehyde as cross-linker. After the immobilization process, I washed nanoparticles four times and measured the protein amount of washing water with both Lowry and Bradford methods, with BSA as standard, but it is more than the protein amount of initial enzyme I added and the protein loading is negative. I calculated protein this way:
protein loading(%)= protein immobilized onto nanoparticles / amount of protein used*100
Enzyme immobilized onto nanoparticles (mg) = amount of protein used (mg) – the amount of protein in washing water (mg)
total protein of washing water (mg)= protein (mg/ml)* the volume of washing water (ml)
First of all I would make the calibration for Lowry or Bradford using cellulase itself, not BSA. The slopes may differ a lot for different proteins. Secondly, the enzyme is diluted strongly in the washings, which may be a source of error, too. Consider to concentrate the enzyme in the washings using ultrafiltration membrane, to the volume of starting reaction mixture and then measure both protein concentration and enzyme activity. Finally make sure that no glutaraldehyde or tannin is leaking from the carrier. These may affect protein estimation, at least with Bradford.
A. E. Ivanov thanks for helping.
before immobilization, I use the ultrasonic bath to disperse nanoparticles and to prevent glutaraldehyde from leaking in immobilization solution, first, I stir glutaraldehyde with nanoparticles for 2 hours and then wash it off. after that, I add the enzyme and stir it overnight to be immobilized. but at last, the washing water's color is clear light brown and I think it shows that something is wrong.
The color may come from Fe3O4 very finely dispersed in colloid form in the solution. You may run "immobilization" without enzyme and see if the color is still in the washings and if it affects your calibration. Another source of color may be tannin or Schiff base formed between GA and the enzyme. Attachment of the enzyme may partially solubilize tannin (I understand, it is physically adsorbed).
A. E. Ivanov I ran immobilization without enzyme. the color of the supernatant with and without glutaraldehyde was light brown and also, I performed protein lowry assay for both of them (without enzyme) and it showed about 6 mg protein. so it is clear that they affect the protein results but I don't know how to remove these errors.
Good, now you see where the error comes from. After GA treatment, try to wash out GA for longer time, like you did during "immobilization" , until you get colorless washings and insignificant Lowry reaction. To prevent oxidation of GA by oxygen during washing, use degassed water.
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