There is a problem i am getting during the immobilization of protein on support . Initially 1-2mg/mL of protein concentration and 1 mg of support is being taken. After immobilization the protein in the supernatant and washing is coming out to be negligible as compared to the initial protein content taken. I have done bradford, UV vis. lowry estimations both are giving results that are not detectable and negligible.
Is there any other method for detection of proteins?
Where are you immobilizing the protein? Is it physical or chemical immobilization? What are you using for washing?
Immobilization of protein was done on multiwalled carbon nanotubes via covalent approach. Phosphate buffer is used for the washing.
I suspect that hydrophobic interactions are ocurring, thus you do not see free protein. You need to wash with a mild detergent solution (do not know if it is possible in your system) to remove protein that is physically adsorbed. To validate my hypothesis, add the protein without reagents for covalent bonding and you should see a similar result, no protein in supernatant.
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