I have a sample with a volume of less than 10 mL, and a fairly low concentration of 24 mg. I want to do a peptide cut-off, but i'm worried that my cut-off results will get poor results. What can i do ?
Really appreciate any help you can provide.
Hello Andi; what do you mean "with doing a peptide cut-off"? What is your procedure for this?
Please look at the following below links which may help you in your analysis:
https://assets.thermofisher.com/TFS-Assets/CMD/Technical-Notes/tn-50-amino-acid-peptides-lpn1197-en.pdf
Thanks
Basic peptides (number of basic amino acids including the N-terminal amino group larger than the number of acidic amino acids including the C-terminal carboxyl moiety) may be dissolved in a small amount of an acidic solvent such as acetic acid or trifluoroacetic acid and then diluted to the desired concentration. However, the safest diluent is PBS at pH 7.0 - 7.4 provided that a concentration of ≤1 mg/mL is sufficient. If delivered as trifluoroacetates, peptides containing a relatively large proportion of Arg and Lys residues tend to be soluble at neutral pH.
See page 14 in the Bachem paper: https://www.bachem.com/fileadmin/user_upload/pdf/Catalogs_Brochures/Bachem_Peptide_Guide.pdf
I have a peptide solution. I will do peptide ultrafiltration / cut-off using MWCO into several sizes 3-5kDa,> 5-10kDa, and> 10kDa to obtain the liophilyz. but I am afraid the results obtained are not good because the peptide volume
Hello Andi Gunawan ;
Thank you very much for clarifying your question. I think it all will depend on your method of peptide analysis; it would be good to know what is your method of peptide analysis and what is the concentration sensitivity of such method.
Once you have this information, if the sensititvity of your analytical method allows it, I would think of diluting your original solution so that you have more sample to carry out your experiment. This is because your current solution volume is low indeed, but your initial peptide amount may be not as low as you are suggesting; this will depend on the sensitivty of your analytical method. This is also relevant because if you will do an important experiment of your research work you will need to carry out triplicate tests for each molecular weight cut-off, at least.
After this, I would do serial dilutions of your original solution and measure the peptide concentration; with this you may obtain a calibration curve, but also and more importantly, the range of concentrations where you can obtain reliable peptide concentration measurements. The next step, is to determine the appropriate initial solution concentration for the design of your molecular weight cut-off experiment.
Hope this helps. Regards.
Andi Gunawan I think that the separation of peptides with so similar masses will be challenging using ultracentrifugation. As the filters are usually designed to retain the analyte, and not to deplete it, your 3-5 kDa fraction will most likely contain peptides of
Thank you, everyone. Isam Eldin Hussein Elgailani Andrei Blasko Rogelio Valadez-Blanco Marta Grzeski
Your answer is very helpfull
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides