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Protein Concentration Koncentracija proteina

Protein Determination by UV Absorption Određivanje proteina po UV Absorption

Near UV Absorbance Kraj UV Absorbance

Near UV Absorbance is UV Absorbance at 280 nm: A simple spectrometer can quantify the amount of protein in a solution.  Radiation absorption by proteins in the near UV depends on the content of Tyr and Tr (and to an extent on the amount of Phe and disulfide bonds). Kraj Absorbance UV je UV Absorbance at 280 nm: A jednostavan spektrometar mogu kvantificirati iznos proteina u rješenje. Apsorpcija zračenja po proteina u blizini UV ovisi o sadržaju Tyr i TR (u omjeru i na količinu i Phe disulfide obveznice). Thus the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5–1.5). Tako A280 varira uvelike između različitih proteina (za 1 mg / mL rješenje, od 0 do 4 [za neke tirozinska-bogata vune proteini], iako najviše vrijednosti su u rasponu 0,5-1,5). This method has its advantages; it is simple, and the sample is recoverable. Ova metoda ima svoje prednosti, ona je jednostavna, a uzorak je ispravljiva. Be that as it may it also has disadvantages which include, interference from other chromophores, and the specific absorption value for a given protein must be determined. Se kao da je svibanj ona također ima nedostataka koji uključuju, interferencija iz drugih chromophores, i specifičnih vrijednosti apsorpcije za određeni protein mora biti određena. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption. The extinction of nucleic acid u 280-nm regiji svibanj biti koliko puta da je od 10 proteina u njihovoj isti valna duljina, i stoga, nekoliko posto nucleic acid može uvelike utjecati na apsorpciju.

Far UV Absorbance Far UV Absorbance

Absorption of the peptide bond is strongly in the far UV with a maximum at about 190 nm   The strong absorption of proteins at these wavelengths has been used in protein determination. Apsorpcija, peptida bond je jako daleko u UV s najviše na oko 190 nm The strong apsorpcija proteina na ovim wavelengths je korišten u protein determination. Because of the difficulties caused by absorption by oxygen and the low output of conventional spectrophotometers at this wavelength, measurements are more conveniently made at 205 nm, where the absorbance is about half that at 190 nm. S obzirom na poteškoće uzrokovane oxygen absorption by a low output konvencionalnog spectrophotometers u ovom valna duljina, mjerenja su jednostavnije made at 205 nm, gdje je oko pola absorbance at 190 nm da. Most proteins have extinction coefficients at 205 nm for a 1 mg/mL solution of 30–35 and between 20 and 24 at 210 nm.  Various side chains, including those of Trp, Phe, Tyr, His, Cys, Met, and Arg (in that descending order), make contributions to the A205. Većina proteina imaju extinction koeficijenti na 205 nm za 1 mg / mL rješenje od 30-35 i između 20 i 24 na 210 nm. Razno strani lanci, uključujući i one TRP, Phe, Tyr, Njegova, Cys, Ispunjeni i ARG ( u tom opadajući red), čine doprinosa na A205. The advantages of this method include simplicity and sensitivity. Prednosti ove metode uključuju jednostavnost i osjetljivost. The sample is recoverable and in addition there is little variation in response between different proteins, thus permitting near-absolute determination of protein. Uzorak je ispravljiva i pored se nalazi malo varijacije u odgovoru između različitih proteina, tako blizu odobre-apsolutni određivanje proteina. Disadvantages of this method include the necessity for accurate calibration of the spectrophotometer in the far UV. Nedostaci ove metode uključuju potrebu za precizne kalibracije je u daleko spektrofotometar UV. Many buffers and other components, such as heme or pyridoxal groups, absorb strongly in this region. Mnogi buffers i druge komponente, kao što su heme ili piridoksal grupe, snažno uvući u ovoj regiji.

See also our Protein Concentration Protocol and Protein Concentration Protocols from other labs . Pogledajte i našu Protokol Koncentracija proteina i protein Koncentracija Protokola iz druge učionice.

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