Hello to everyone.
My work focus on studying PTMs and I have successfully phosphorylate my protein in vitro (Checking the reaction products by SDS-Page, all of my protein should be phosphorylated).
What method do you recommend for determining the molar concentration of my sample?
> Keep in mind that:
a) phosphate group adsorbs at 260nm, so could be difficult to estimate the molar concentration by UV 280nm.
b) my protein has only one aromatic aa, so Bradford or Lowry assay could be not sensitive.
c) one possible solution could be a Biuret assay (that detect the presence of peptide bond).
Do you have anything else to recommend?
Determining the molar concentration of your sample can be a crucial step in further experiments, and I can definitely suggest some methods that might work for your particular case.
Given the limitations you mentioned, a Biuret assay seems like a reasonable option, as it can detect the presence of peptide bonds regardless of the presence of aromatic amino acids. Another possible option could be a colorimetric phosphate assay, which can be specific for the detection of phosphate groups.
One popular method for colorimetric phosphate detection is the Malachite Green assay, which involves the binding of malachite green to the phosphate group and subsequent measurement of absorbance at a specific wavelength. This method can be sensitive and specific for phosphate detection, and it can be adapted for use with small sample volumes.
I would recommend considering both the Biuret assay and a colorimetric phosphate assay such as the Malachite Green assay as potential options for determining the molar concentration of your phosphorylated protein sample. Good luck with your experiments!
The Bradford protein assay detects, in part, the basic amino acids (Lys, Arg), so it should be useful for your protein if it contains a normal complement of such residues. The key to making this, or another, assay accurately is the choice of a suitable standard protein that has a similar amino acid content.
I don't think phosphate absorbs at 280 nm. Even if it does, you can subtract the background from the buffer.
Dear Sebastian, you are in righ! After phosphorylation I plan to perform another PTM on my sample, subsequently I will procede with several experiments where is necessary to known the samples concentration
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence