Hi,
I am having a 4kDa peptide expressed from E. coli and purified. I would like to quantify the concentration before I use it for crystallization experiment. Unfortunately, it does not contain any W or Y. I learned about OD214 on the Web, but I am not sure how could I compute its extinction coefficient. Or what kind of standard should I use so that I can construct the standard curve?
Thanks.
You could use primary amino groups for quantification by reacting with ninhydrin, fluorescamine or OPA, for example, as long as your purified peptide is not in a buffer containing amino groups. Use something with one amino group as a standard (e.g. Tris or ethanolamine).
For such a small peptide, it would be practical to have it chemically synthesized.
Hi Sam, just to add to Adam's suggestion, the E coli peptide will probably have a blocked N-terminus, so it has to have some Lys residues in order to quantify it using amine reactive reagents.
Alternatively, you could try reagents that measure peptide bonds
http://www.piercenet.com/method/chemistry-protein-assays
Whatever method is used, you will need a standard curve. The standard substance must be chosen to be chemically similar, with respect to the detection reaction, as the peptide. People usually use albumin as a standard in protein assays, but in this case it would not be representative. For the biuret assay, you would need a peptide of the same length, similar composition, and a known concentration.
If you have a lot of the purified peptide, you could use gravimetry to quantify it, i.e. weigh it. You would have to get rid of all the solvent, buffers, and salts, and freeze dry it. If the peptide is delivered in a volatile buffer, such as ammonium bicarbonate or ammonium formate, the buffer can be removed at the same time as the water by lyophilization.
Hi Sam, if you are going to use the BCA assay to measure peptide bonds, then lysozyme is too big (MW ~14kDa).
Aprotinin (~6kDa) would be a good choice, but it is very basic. Insulin (~5.8 kDa) could also be a contender.
But, coming back to Adam's comment, if you are doing crystallization of this peptide, you will need a lot of it. You say that you don't have much of it. Therefore, synthesizing the peptide will probably be more practical for your experiments.
To previous suggestions I would add this. The proteins, or better the peptide bond, absorb light also in the low wavelength range. In general the range between 205 and 210 nm is well suited for the calculation of an extinction coefficient at 280 nm. I can suggest some old semi-empirical relationships but sufficiently accurate. The extinction coefficient at 280 nm can be obtained from absorption measurements at 205 and 210 nm and 280 nm on the same sample. E1cmmg/mL values of 32.8 ± 1.6 and 21.6 ± 1.7 can be used, respectively, at 205 and 210 nm. These are average values obtained on a group of seven well characterized proteins. In general the extinction coefficients at 210 nm range from 20 to 24. The protein solution must be dilute for the A 280 assay because proteins absorb strongly at 205 nm, and at this wavelength there is less variability from protein to protein. At this wavelength stray light can be a major problem and a 10 micrograms/microliter solution of bovine serum albumin can be used as a standard to minimize the stray light. Make a dilute albumin solution between 0 and 10, for instance a solution of 5 micrograms/microliter can be easily prepared diluting ½. Hence you can determine the concentration of the unknown (the measurement must correspond to a concentration between 0 and 10 micrograms/microliter) by interpolation using a buffer blank as zero absorbance. This is acceptable because in the 0 to 10 microgram/microliter range the relationship of absorbance and concentration is linear. Use bi-distilled water for solutions.
Moreover, the following empirical equation can also be used:
E 1mg/mL at 205 nm = 27.0 + 120 x(A280nm / A205nm)
Or even:
Protein concentration (mg/mL) = (Absorbance at 205 nm)/31.
Good experiment.
Hi,
How do you know you dont have much of the peptide? You will only know when you can measure its absorbance. Does your peptide have cysteine residues?
Hello, filter your peptide (4k) on a filter device (cutoff 1k) and then using the filtrate as blank solution, take the spectrum (retentate) between 190-300 nm. You can evaluate the 205 nm extinction coeff here: http://nickanthis.com/tools/a205.html
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