During the study of protein degradation, Substrate and product quantification using conventional methods like Bradford , Lowry etc. could not applied because they are based on the reaction of added chemicals with amino acid having Benzene ring in their structure. And also the bacteria which are responsible for protein degradation release enzymes for protein degradation. These enzymes are also proteins.
Although there is qualitative assays are in literature but quantification of protein degradation with respect to growth kinetics and enzyme kinetics are lacking. So any one can suggest me How can quantification of bacterial protein degradation possible?
Do you want to quantify degradation of a specific protein or a general mixture?
If it's single (or few) specific proteins you could label your protein with biotin or fluorescent label.
Then after degradation experiment run the SDS-PAGE and then visualize your labelled protein with fluorescent imager.
Or in case of biotin labeling detect your proteins with western blot.
You could quantify protein degradation by comparing bands after degradation to control using software like ImageJ.
Good luck.
T.
Which degradation assay?
Is that protein hydrolysis by enzymes? If yes, u can check it by doing SDS-PAGE profile of degraded and undegraded samples.
If u want to check presence of enzymes digesting proteins, you can check by zymogram assay.
If you want to quantify the digestion of protein, it can be done by TCA precipitation method where you will have to quantify the protein content in TCA-soluble fraction and the sample (without the addition of TCA) by Lowry's method. Increase in protein content in TCA soluble fraction suggests degradation.
It isn't clear to me what your experimental system is. Are you trying to measure the degradation of an external source of protein by bacteria? If so, are you adding one specific protein, or are you using a heterogeneous source of protein? What happens to the protein when it is degraded? Is it reduced to small peptides and amino acids? Are the products taken up by the bacteria? Can you separate the bacteria from the protein at the end of the incubation?
One possibility for a crude system is to measure the amount of acid-precipitable protein left in the sample, once the bacteria are separated from the sample.
A very common method for following protein degradation in some food products is quantifying total nitrogen, peptide nitrogen and amino acid nitrogen separately (or even just total nitrogen and non-protein nitrogen): it will give you an idea of how intact are the proteins and how intense is the degradation. The quantification of nitrogen is through Kjeldahl.
https://www.researchgate.net/publication/43072852_Influence_of_pre-cure_freezing_of_Iberian_ham_on_proteolytic_changes_throughout_the_ripening_process
Article Influence of pre-cure freezing of Iberian ham on proteolytic...
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules