First, blank the spectrophotometer. You do this with a solution that has ALL of the components that you would expect to find in your sample EXCEPT the molecule of interest. So, if you want to measure antibody that is in PBS/EDTA your blank has to be PBS/EDTA.

However, you should also consider how the sample has been prepared. If you desalted the antibody into the PBS/EDTA buffer, the blank is the column equilibration buffer. If you dialysed the Ab into buffer, the strictly correct blank to use is the spent dialysis buffer (not fresh buffer). If you diluted 10mg/ml Ab in 50 mM Tris/0.1 % azide into PBS/EDTA to give a 1mg/ml Ab solution, the strictly correct blank to use then is a 1/10 dilution of the tris/azide buffer in the PBS/EDTA buffer, so that the ONLY difference between the blank and sample is the presence of the molecule that you wish to measure.

Finally, you will also need to use a cuvette that can transmit UV wavelengths e.g., quartz.

The overlap of the spectra of fullerenol and proteins might cause some trouble and require measurements at several wavelength and some deconvolution. Try to model your expectations and then factor in the usual uncertainties and measurement errors to check how robust and sensitive your setup is.

Are you coupling the antibody to the Fullerenol using SMCC? How well does that work? Are you aware of fullerene derivatives with grafted malonic acid, where you have carboxylic groups available that may be activated directly with carbodiimide chemistry (e.g. EDC/NHS)?

Em Husein What is the point of posting "answers" generated by a Chatbot? This won't advance science any further, and, it won't help the OP.

When feeding the question into ChatGPT I get exactly this:

"When working with UV-VIS spectroscopy, selecting the appropriate baseline and reference solution is crucial for accurate characterization of your samples. Here are some recommendations for your specific case:

Baseline: The baseline is the spectrum obtained from the solvent or buffer alone, without any sample. It helps to identify and subtract the contribution of the solvent or buffer to the overall spectrum. In your case, using water as the baseline is generally acceptable, especially if the solvent or buffer used in your samples is primarily water-based. However, keep in mind that if your samples contain components that significantly absorb in the UV-VIS range, it might be better to use the buffer alone (PBS-EDTA) as the baseline.

Reference: The reference solution is used to calibrate and normalize the spectra. It is typically a solution that is optically transparent and does not contribute any significant absorption or scattering. In your case, using a clear cuvette as the reference is appropriate, as long as the cuvette material is transparent in the UV-VIS range and does not introduce any spectral features.

Alternatively, you can also use the same buffer (PBS-EDTA) as the reference solution. This ensures that any variations or artifacts introduced by the cuvette material are accounted for in both the reference and sample measurements.

Procedure:

By following these steps, you should be able to obtain more distinct and meaningful spectra for your molecules of interest while accounting for the contributions of the solvent or buffer."