I am using a peptide and have faced challenges in preparing a transparent peptide solution. My goal is to prepare a peptide solution in MES buffer and then use the solution to immobilize the peptide on the nanoparticle's surface using EDC/NHS chemistry in MES buffer (pH 5-6).
Since the peptide has poor solubility in water and considering the desired pH for the conjugation reaction: I cannot use acids to dissolve the peptide as this would lower the pH of the solution. I tried using 20% acetonitrile, but the solution was not transparent and contained many insoluble particles. With 10% DMSO, I achieved a mostly transparent solution, but a few tiny insoluble particles remained.
Another issue is determining the peptide content indirectly by measuring the remaining peptide in the supernatant. Using HPLC at a wavelength of 220 nm, I observed a wide peak that seems to be related to DMSO, not the peptide (Since injecting the blank solvent showed the same peak). Could you please assist me with the following questions:
How can I prepare a clear solution for the peptide in the MES buffer?
How can I easily detect the peptide's absorbance at 220 nm (without any matrix effect from DMSO or MES both have absorbance at 220 nm)?
Does your peptide contain any aromatic amino acids? If so, you can use the absorbance at 280 nm instead of 220 nm. That might make it easier to subtract the background absorbance from the solvent. Make sure you are using quartz cuvettes for far-UV absorbance measurements.
The peptide sequence contains 4 aromatic amino acids out of a total of 12 residues. However, its absorbance at 280 nm is undetectable by HPLC due to a noisy spectrum.
You might be able to improve the aqueous solubility of the peptide using a detergent. If the detergent does not contain any amino or carboxyl groups, it should not prevent the immobilization reaction. Removal of detergents afterwards can be problematic, but if you can use CHAPS detergent, it should be possible to remove it from nanoparticles by dialysis because the micelles are very small.
Dear Houra
It is easy to make a clear stock solution of peptide in urea 6M. Then, the calculated values can be taken from this solution and added to the MES buffer. Finally, a desalting column (e.g., a size exclusion chromatography-based column) can be used to remove urea, MES, EDC and other small molecules.
Good Luck
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