I want to measure the small peptide (2-3 kDa, 25 residues) concentration, but it hasn´t any aromatic residue, so it is not possible to use 280 nm absorbance.
Moreover, Bradford assay did not work, and Lowry assay gave us very noisy results.
Are there another techniques to measure peptide concentration?
Thank you so much.
Please try Wadell’s method. Simple yet reliable!!
Link:
Waddell, W. J. (1956) A simple UV spectrophotometric method for the determination of protein. The Journal of Laboratory and Clinical Medicine,48, 311–314.
How about derivatization or using a different type of detector (e.g. RID, MS).
You can perform aminoacid analysis, or ICP/MS if your peptide contain sulfur atoms. Feel free to contact me if you need more information.
You can detect using a wavelength of 214 nm , which is the zone where the amide group absorbs (peptide bonds).
I think your best (more accurate) option is to do amino acid analysis as Arnaud suggested.
Hi, Carlos.
If your peptide has a tryptophan in sequence, you can measure it with relatively high sensitivity (about 2 times more than for the 280 nm absorption) and high selectivity by the HPLC with fluorimetric detection (excitation 260 nm, emission 295 nm).
P.S.: If i get you right, your peptide hasn't any phenylalanin or tyrosin in backbone, has it?
as somebody else mentioned, measure OD at 214 nm. You will need proper cuvettes transmitting at this wavelength, as well as using water or compatible solvents with low / no absorption at 214 nm. You may want to compare with a reliable standard solution of the same peptide, or draw an estimate of the peptide abosrbance to be expected on the basis of its amino acid composition, using data from: Kuipers and Gruppen, J Agric Food Chem 2007; 55: 5445-51. We have been using the latter for our calculations for a few years, with satisfactory results.
You can measure the concentration of the peptide solution by UV. The amide group, which is present in the peptide absorbs at 210 nm wavelength. So using Beer-Lambert's law, you can measure the concentration.
Hi Carlos,
there are different alternatives depending on what you want
HPLC at 214 did not work for me...I got lots of picks in my peptide mixture, but it will depend on the nature of your sample...you can give it a try concentrating the sample and a normal gradient and see how it goes.
if you only want to quantify the protein content nano-drop could give you a good preliminary estimation without wasting too much sample.
You could also try SDS gel with the software to quantify the width of the band...it gives you an estimation.
If you are interested in determination of all the amino acid contents of your sample I would recommend to perform amino acid analysis and add all the residues that you get from your extract to determine the total amino acid content....this is the most accurate for sure...although it takes time
I hope it helps and good luck
Marco
Hi Carlos,
I am facing the same difficulties you are (were) facing.
I am working with small, 30 - 100 amino acid long, dipeptides. One is (proline-alanine)15
It contains no charge and is fairly small. Can't use Bradford reagent.
Can't "see" it in a gel. It doesn't bind coomassie or silver stain.
Did you ever try the methods of detection suggested in this thread?
Did any of them work? Did you find any other methods helpful?
I would gratefully appreciate any advice or feedback.
Thanks,
Beth
Hello Beth,
Finally, I calculated the concentration of the peptide by measuring Absorbance at 214-220 nm. To obtain the extinction coefficient I measured a sample of the same peptide that I knew the concentration (because I did solubilize a known amount of microgrames).
I also check the approximate concentration by measuring 1H-NMR spectrum.
I hope to be helpful.
Good Luck!
Carlos.
I tried reading RGD using absorbance at 214 nm on a TAKE3 plate and it worked for concentrations 0.005-1 mg/ml!
Thanks,
Olivia
I do have same question how to measure small peptide(5-7 Amino acids) concentration? it do not have any aromatic residue.
You should use amino acid analysis: acidic hydrolysis to obtain amino acids, dericatisation with AccQ-tag reagent, and analysis by UPLC with fluorescence detection.
Alternatively, you can use ICP/MS of your Peptide contain sulfur atoms (cysteine or methionine).
You can contact me if you need more information.
Please try Wadell’s method. Simple yet reliable!!
Link:
Waddell, W. J. (1956) A simple UV spectrophotometric method for the determination of protein. The Journal of Laboratory and Clinical Medicine,48, 311–314.
Cysteines also have absorbance beyond 214-220 range, in addition to cyclic ring side group aa's, and are helpful for measuring concentrations (depending on their redox state)
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides