I want to perform a fluorescence quenching assay of peptide, to confirm if it is a transmembrane domain. The peptide has one tryptophan in the middle of peptide (28 residues). Am planning to do acrylamide/ KI quenching to study the same. Could anyone tell me what percentage of purity of peptide is required for fluorescence quenching assay?
Judging by the size of your peptide, you can purify the sample via preparative HPLC on a diphenyl column using a linear gradient of water and 90% acetonitrile. Acidification of the mobile phase with 1% HOAc or 0.1% TFA may also increase peptide resolution, thus giving higher purity. This is a general protocol. In most cases, after 1 HPLC purification, I would have 95%-99% pure peptide.
Thanks for your answers.
I have got a peptide with > 95 % purity and it is a transmembrane region of a protein. It has 2 phenylalanine and 2 trysoine with no tryptophan, the sequence is as follows : KKKIFIYILSIIYLSVCVMNKIFAKKKK. The lysine residues are added at the end to increase the solubility of the peptide. I want to confirm that the peptide is a membrane protein, by performing quenching assays.
In some papers, they have done quenching assay using acrylamide, with phenylalanine and no tryptophan. I also have tried the same with phenylalanine excitation at 357 nm and emission from 270 to 450 nm. When plotting the graph between F0/F versus [Q] concentration and am getting a upward curvature in graph.
When reading regarding this, I came to know that it may be because of combined static and dynamic quenching. Some said that it may be because of interference. And i don't know how to calculate the Ksv and Ka value separately.
If possible, please help me in solving this. I would be so thankful for your help.
Also, some suggested that i have change a tyrosine to tryptophan in the middle and then do quenching assay.
If you have suggestions regarding this, please do help me. Thanks in advance.
Sorry. The excitation for phenylalanine used was 257 nm. I read that article and corrected for the inner filter effect of absorption using the formula given and plotted graph between F0/F corrected value versus acrylamide concentration.
But, when am getting a downward curvature and not straight line. Herewith, am attaching a file in which two graphs are there: first one without correction in the presence and absence of SDS micelles and second one with correction in the presence and absence of SDS micelles.
Could you please have a look into the file and help me regarding the same?
Thanks in advance.
Thank you so much for your valuable suggestions.
I used 4 M acrylamide stock concentration. From the stock, i used 1 ul for 2 ml sample volume, constituting buffer and peptide and took the reading. To this, i add again 1 ul of acrylamide, till the fluorescence is quenched. The fluorescence observed maximum at each acrylmide concentration is taken as F and fluorescence without acrylamide is taken as F0. Then, I plotted graph F0/F versus [Q], and got an upward curvature.
When I was taking high concentration of peptide, the peak is going beyond 1000 units. So, I took low concentration of peptide approximately 1 micromolar. In papers regarding peptide characterization, they have taken low quantity 1-3 micromolar concentration, for quenching assay and the acrylamide concentration is also low only.
I tried with KI quencher also. I took 4M KI and when am adding to the same volume and concentration of peptide, it quenched with 1-3 ul of 4M KI.
I have gone through the paper and will do the necessary modifications for my experiments. I will keep in touch you, for any further queries regarding the same.
Thank you for your kind help.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology