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Experiment design to study amino acid and protein interactions?
- Charlesmsa7
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5 years 2 months ago - 5 years 2 months ago #1
by Charlesmsa7
Experiment design to study amino acid and protein interactions? was created by Charlesmsa7
Hello everyone,
Greetings!
I have few clarifications on setting up the experiment to study amino acids - protein interactions?.
I want to study how my protein (MW - 18 kDa, concentration - 5mg/ml, pretty stable at room temperature also) binds with all 20 amino acids. In this case How much i should immobilize on the surface (I have CM5 chip). Its known the interaction between and amino acids make transient interactions with their receptor proteins. In this case how i should set up the assay? just binding alone or detailed kinetic assay?. Also how many concentration of amino acids I should use. I would like to know which amino acid has more affinity/ or binds well with the receptor.
Thank you.!
Greetings!
I have few clarifications on setting up the experiment to study amino acids - protein interactions?.
I want to study how my protein (MW - 18 kDa, concentration - 5mg/ml, pretty stable at room temperature also) binds with all 20 amino acids. In this case How much i should immobilize on the surface (I have CM5 chip). Its known the interaction between and amino acids make transient interactions with their receptor proteins. In this case how i should set up the assay? just binding alone or detailed kinetic assay?. Also how many concentration of amino acids I should use. I would like to know which amino acid has more affinity/ or binds well with the receptor.
Thank you.!
Last edit: 5 years 2 months ago by Charlesmsa7.
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- Arnoud
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5 years 2 months ago #2
by Arnoud
Replied by Arnoud on topic Experiment design to study amino acid and protein interactions?
You can calculate the Rmax of the reaction with the formulas at
www.sprpages.nl/immobilization/immobilization-procedures
.
Normally we aim for 100 RU but if you are unsure of the activity of the protein immobilize more or immobilize two or three concentrations giving a theoretical Rmax of 50, 250 and 2500 RU. Always immobilize the highest at channel 4.
If you really want to compare dufferent amino acids you should do the full kinetics.
Best way to determine the full kinetics is a six step concentration range with a 3x dilution series. You have to figure out yourself where to begin but the final concentration range should be between 0.1 and 10 xKD of the interaction.
When you run the series overnight you have 15-18 hours of measuring time!
Kind regard
Arnoud
Normally we aim for 100 RU but if you are unsure of the activity of the protein immobilize more or immobilize two or three concentrations giving a theoretical Rmax of 50, 250 and 2500 RU. Always immobilize the highest at channel 4.
If you really want to compare dufferent amino acids you should do the full kinetics.
Best way to determine the full kinetics is a six step concentration range with a 3x dilution series. You have to figure out yourself where to begin but the final concentration range should be between 0.1 and 10 xKD of the interaction.
When you run the series overnight you have 15-18 hours of measuring time!
Kind regard
Arnoud
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