Determination of Protein Concentration by Uv Absorption

Determination of Protein Concentration by Uv assimilationHighly susceptible to defilement by minces, biological materials and saltsProtein amino harsh composition is extremely important, thus the choice of a banal is real difficult, especially for purified proteinsAbsorbance is heavily influence by pH and ionic ability of the solution.General ConsiderationsThis is often roled to visualise protein soaking up prior to a more(prenominal) sensitive rule acting so the protein can be diluted to the correct upchuckQuantitative ProcedureZero the spectrophotometer with a modify blankMake a modular curve using your regular of choice in the expected dousing range, using the same cowcatcher that your un enjoyn sample is in. ca-ca the meshance values at 280 nm in a quartz cuvette ordain sample into quartz cuvette (make sure denseness is in the range of 20 g to 3 mg defer absorbance at 280 nmEstimation ProcedureZero spectrophotometer to urine (or buffer)Take the absorbance at 280 nm in a quartz cuvetteChange wavelength to 260 nm and zero with water (or buffer)Take absorption at 260 nm in a quartz cuvetteUse the following equation to estimate the protein concentrationProtein (mg/mL) = 1.55*A280 0.76*A260DiscussionDetermination of protein concentration by ultraviolet absorption (260 to 280 nm) depends on the presence of aromatic amino acids in proteins. Tyrosine and tryptophane absorb at approximately 280 nm. Higher orders of protein structure also may absorb UV light or modify the zep absorptivities of tyrosine and tryptophan and thus the UV detection is highly sensitive to pH and ionic strength at which measurement is taken. Many opposite cellular components, and particularly nucleic acids, also absorb UV light. The ratio of A280/A260 is often used as a measurement of the purity of protein or nucleic acid samples during their purification. The real advantages of this method of determining protein concentration are that the sample is not destroyed an d that it is very rapid. Although contrasting proteins leave alone contract different amino acid compositions and thus different molar absorptivities, this method can be very accurate when comparing different solutions of the same protein.To make an accurate determination of protein concentration, you leave suck to erect a standard curve (A280) with known amounts of purified protein. You leave alone also have to provide a blank that is appropriate for the sample and contains the same concentrations of buffer and salts as the sample. It is often convenient to dialyze the sample and measure the absorbance of the retentate (still in the dialysis sack) using the dialysate as the blank. Care must be taken to use quartz cuvettes, since glass absorbs UV light. A handy equation to estimate protein concentration that is often used isProtein (mg/mL) = 1.55*A280 0.76*A260However, it is also a good judgement to always use a standard curve and suggested that you evaluate the symmetrica lness of the results using the above equation with results using a standard curve.This method is the least sensitive of the methods discussed here. For increased sensitivity, the wavelength can be lowered to the range of 210 to 225 nm. This measures the amide bond in proteins. However it is much more subject to incumbrance from more more biological components and compounds used to make buffer solutions.If you dont know what the protein concentration of an unidentified sample is likely to be, the ultraviolet method king be a good starting point. Prepare a standard curve for the absorbance at 280 and 260 nm. After you have the data for the standard curve, rezero the spectrophotometer with water. Place your samples into a dry 1 mL quartz cuvette and read the absorbance. If the A280 of your unknown sample is less than 2, you should probably not dilute your sample further. If the absorbance is 2, dilution will be required. When you are finished with the firstly measurement, the unkn own can be returned to its sea captain tube with minimal loss.The Lowry AssayReferencesO.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) J. Biol. Chem. 193 265. (The original method)Hartree E. E. (1972). Anal. Biochem. 48 422 (This registration makes the baulk one-dimensional over a larger range than the original stay)J.R. Dulley and P.A. Grieve (1975) Anal. Biochem. 64 136. (This is a useful modification of the original Lowry method that includes 0.5% sodium dodecylsulfate in the alkaline reagent. This obviates interference from many detergents and helps disperse membranes in the sample.)A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70 241. (Another useful modification of the original Lowry method that can be useful when the solution contains interfering contaminants. The proteins in the samples are precipitated by a mixture of sodium deoxycholate and trichloroacetic acid and centrifugation prior to canvas. If the contaminants stay in the supernatant they c an be removed and the amount of precipitated protein regardd.Quick GuideHow does it attain?The first step is a Biuret reception which reduces Cu+2 to Cu+1The second reaction uses Cu+1 to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). This is perceptible in the range of 500 to 750 nmDetection Limitations2- speed of light gAdvantages raw(a) over a wide rangeThe most commonly referenced procedure for protein determinationCan be performed at room temperature10-20 multiplication more sensitive than UV detectionCan be performed in a microplate formatDisadvantagesMany substances interfere with the chipAlkaline hair reagent is laborious to prepare and will develop carbonate scales over reposition which interfere with optical activity, thus it must be prepared good dailyTakes a considerable amount of time to performThe assay is photosensitive, so illumination during the assay must be kept coherent for all samplesAmount of color varies with different prot einsGeneral ConsiderationsSome researchers have reported that repeated assays in the same cuvettes cause them to be printMany chemical distri neverthelessors sell a modified Lowry assay that is more stable and sensitive than homemade versionsSince trim copper is detected in the procedure, make sure that the distilled water used in the procedure is provide from plastic lines and not copper lines. In general water from 18 megaohm water polishers is satisfactoryVariation in the content of tyrosine and tryptophan residues will influence the assayProcedureAlkaline Reagent0.1 M NaOH2% Na2CO30.5% Na Tartrate (use of thou salt will cause SDS to be insoluble)0.5% Na Dodecylsulfate papal bull Reagent1% CuSO4*5H2OAssay aggregate (MAKE FRESH EACH DAY)50 mL alkaline reagent and 0.5 mL copper reagentFolin-Ciocalteu ReagentDilute with an equal peck of water to prepare the desired volumeProcedure enlarge samples containing up to 100 g of proteinBring all tubes to 1 mL total volume with water. Be sure to have two tubes containing only water as blanks. Also use reagent or buffer blanks if needed.Prepare the Assay Mix and diluted Folin-Ciocalteu reagent.To each tube add 5 mL of assay mix and thoroughly vortex.Incubate tubes at room temperature for 10 min.Add 0.5 mL of diluted Folin-Ciocalteu reagent. Vortex immediately.Incubate at room temperature for 30 min.Vortex the tubes, zero the spectrophotometer with the blank and measure absorbance at 660 nm (or other appropriate wavelength). The data from the standard curve are usually linear enough that a straight-line interpolation can be used to determine the concentration of unknowns.DiscussionThe Lowry method relies on two different reactions. The first is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a Biuret chromophore. The second is the reduction of Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and tryptophan residues. The reduced Folin-Ciocalteu reagent is blue and thus detectable with a spectrophotometer in the range of 500-750 nm. The Biuret reaction itself is not all that sensitive. Using the Folin-Ciocalteu reagent to detect reduced copper makes the assay nearly 100 times more sensitive than the Biuret reaction alone.The assay is relatively sensitive, but takes more time than other assays and is susceptible to many interfering compounds. The following substances are known to interfere with the Lowry assay detergents, carbohydrates, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl compounds, disulfide compounds, magnesium and calcium. Most of these interfering substances are commonly used in buffers for preparing proteins. This is one of the major limitations of the assay. The Lowry assay is sensitive to variations in the content of tyrosine and tryptophan residues. If the protein you are assaying has an unusual content of these residues, an appropriate substitute standard is requir ed. The standard curve is linear in the 1 to 100 ug protein region. The absorbance can be read in the region of 500 to 750 nm. Most researchers use 660 nm, but other wavelengths also work and may reduce the effects of contamination (e.g. chlorophyll in plant samples interferes at 660 nm, but not at 750 nm).