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Protein Determination by UV Absorption Protocol Protein Bestemmelse ved UV Absorption protokol

Protein Determination by UV Absorption Protocol, ( Modified from the protocol by Alastair Aitken and Michèle P. Learmonth) Protein Bestemmelse ved UV Absorption protokol, (ændret fra protokollen af Alastair Aitken og Michèle s. Learmonth)

Materials for UV Protein Concentration Materialer til UV Protein Koncentration

1. 0.1 M K2SO4 (pH 7.0). 0,1 M K2SO4 (pH 7.0).
2. 5 mM potassium phosphate buffer, pH 7.0. 5 mm kalium fosfatbuffer, pH 7,0.
3. Nonionic detergent (0.01% Brij 35) Nonionic vaskemiddel (0,01% Brij 35)
4. Guanidinium-HCl.
5. 0.2-µm Millipore (Watford, UK) filter. 0,2-μ m Millipore (Watford, UK) filter.
6. UV-visible spectrometer: The hydrogen lamp should be selected for maximum     intensity UV-synlige spektrometer: brint lampe bør udvælges for maksimal intensitet
at the particular wavelength. på de særlige bølgelængde.
7. Cuvets, quartz, for <215 nm. Cuvets, kvarts, for <215 nm.

Protein Concentration UV Method Protein Koncentration UV Metode

Estimation of Protein by Near UV Absorbance (280 nm): Estimering af Protein ved Nær UV absorptionen (280 nm):

1. A reliable spectrophotometer is necessary. En pålidelig spektrofotometer er nødvendig. The protein solution must be diluted in the Proteinindholdet opløsning skal fortyndes i
buffer to a concentration that is well within the accurate range of the instrument (see Notes 1 and 2). buffer til en fusion, som er godt inden for den nøjagtige rækkevidde af instrumentet (se note 1 og 2).

2. The protein solution to be measured can be in a wide range of buffers, so it is usually no problem to find one that is appropriate for the protein which may already be in a particular buffer required for a purification step or assay for enzyme activity, for example (see Notes 3 and 4). Proteinindholdet løsning, der skal måles, kan være i en bred vifte af buffere, så det er normalt ikke noget problem at finde en, der er relevante for det protein, som allerede kan være i en særlig buffer kræves til en rensning skridt eller assay for enzym-aktivitet, for eksempel (se note 3 og 4).

3. Measure the absorbance of the protein solution at 280 nm, using quartz cuvets or cuvets that are known to be transparent to this wavelength, filled with a volume of solution sufficient to cover the aperture through which the light beam passes. Mål absorbansen af proteinet opløsning på 280 nm, ved hjælp af kvarts cuvets eller cuvets, der vides at være gennemsigtig til denne bølgelængde, fyldt med et volumen opløsning tilstrækkelige til at dække hullet gennem hvilke lysstrålen passerer.

4. The value obtained will depend on the path length of the cuvet. Den fundne vaerdi vil afhænge af stien længden af cuvet. If not 1 cm, it must be Hvis det ikke er 1 cm, skal det være
adjusted by the appropriate factor. justeret med den relevante faktor. The Beer-Lambert law states that: A (absorbance) = ε cl where ε = extinction coefficient, c = concentration in mol/L and l = optical path length in cm. De Beer-Lambert lov hedder det: A (absorbansen) = ε cl, hvor ε = udslettelse koefficient, c = koncentration i mol / L og L = optisk sti længde i cm. Therefore, if ε is known, measurement of A gives the concentration directly, ε is Så hvis ε er kendt, måling af A giver fusionen direkte, ε er
normally quoted for a 1-cm path length. normalt citeret for en 1 cm sti længde.

5. The actual value of UV absorbance for a given protein must be determined by some absolute method, eg, calculated from the amino acid composition, which can be determined by amino acid analysis. Den faktiske værdi af UV absorbansen for et bestemt protein skal bestemmes af nogle absolutte metode, f.eks beregnet ud fra den aminosyre sammensætning, som kan bestemmes ved aminosyre analyse. The UV absorbance for a protein is then calculated according to the following formula: A280 (1 mg/mL) = (5690nw + 1280ny + 120nc)/M UV absorbansen for et protein er derefter beregnet efter følgende formel: A280 (1 mg / ml) = (5690nw + 1280ny + 120nc) / M
where nw, ny, and nc are the numbers of Trp, Tyr, and Cys residues in the polypeptide of hvor nw, NY, og NC er antallet af TRP, tyr, og Cys reststoffer i polypeptider af
mass M and 5690, 1280 and 120 are the respective extinction coefficients for these residues (see Note 5). masse M og 5690, 1280 og 120 er de respektive udslettelse koefficienterne for disse restprodukter (se note 5).

See also Protein Concentration Protocols from other labs . Se også Protein Koncentration protokollerne fra andre labs.

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Protein Identification Protein Identification

Recombinant Protein Expression Systems Rekombinant Protein Expression Systems

Protein Expression Techniques Protein Expression Teknikker