Hi everyone,
does someone have any experience in calculating the degree of labelling of TAMRA labelled protein?
I have found this useful link,
https://www.thermofisher.com/cz/en/home/references/molecular-probes-the-handbook/fluorophores-and-their-amine-reactive-derivatives/long-wavelength-rhodamines-texas-red-dyes-and-qsy-quenchers.html#head1
though it specifies it is very complicated to quantify the degree of labelling.
My protein has been labelled through cysteines with tamra maleimide dye. I usually calculate the concentration of the protein by using its absorbance at 280 nm, and subtracting the contribution of the dye (using the correction factor found in literature- 0.36 - and its absorbance at the maximum). The dye attached to the protein has a different spectrum compared to the unbound dye (see link above), thus I am not sure which one I should consider as a maximum in the range of absorption of the day (500-600nm).
I hope someone could share his/her experience with me. Thank you very much in advance.
Federica
Generally,three main factors should be considered before choosing a method.
1-Sensitivity- A method is known to be sensitive if it can detect protein at very low concentrations.
2-Specificity- Specificity of a method is determined by its efficiency to detect protein specifically from all other interfering substances.
3-Time- Duration of time for assay completion and interpretation of results.
Therefore, different methods of estimation of Protein concentration,such as are mentioned below:
*Biuret method.
*UV absorption.
*BCA assay.
*Lowry assay.
*Bradford assay.
However, quickest way to estimate the amount of protein in solution is to use UV-vis to measure absorbance directly, but this is generally not very accurate or sensitive. Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.
Another approach is to measure the concentration of the protein using a different method, such as the Bradford or BCA assay. The concentration of TAMRA is still measured by its visible absorbance.
Generally,three main factors should be considered before choosing a method.
1-Sensitivity- A method is known to be sensitive if it can detect protein at very low concentrations.
2-Specificity- Specificity of a method is determined by its efficiency to detect protein specifically from all other interfering substances.
3-Time- Duration of time for assay completion and interpretation of results.
Therefore, different methods of estimation of Protein concentration,such as are mentioned below:
*Biuret method.
*UV absorption.
*BCA assay.
*Lowry assay.
*Bradford assay.
However, quickest way to estimate the amount of protein in solution is to use UV-vis to measure absorbance directly, but this is generally not very accurate or sensitive. Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.
Thank you for your replies. I agree with you that UV-vis spectroscopy is not the most sensitive and accurate method, but I thought I could use it to have a rough idea about the degree of labelling of my TAMRA labelled protein.
To get this information, I need to calculate the concentration of the protein by using its extinction coefficient and its absorbance at 280 nm, subtracted from the contribution of the dye at 280 (using the tabulated correction factor). And also, the concentration of the dye by taking advantage of its absorption in 500-600 nm range. If I am correct, my doubt concerns which wavelength as absorption maximum of the dye to take into account, since when the tamra is bound to the protein, it has a different absorption spectrum compared to the one of the free dye.
Probably due to the changes in the dipole moment, thus the probability and the energy of the transitions are affected.
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