生化分析检测技术课件.ppt

1、12.1.1 吸光光度法vAbsorbance Assay (280 nm)vConsiderations for usevAbsorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is

2、linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. Any non-protein component of the solution that absorbs ultraviolet light will intefere with the assay. Cell and tissue fra

3、ctionation samples often contain insoluble or colored components that interfere. The most common use for the absorbance assay is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. An absorbance assay is reco

4、mmended for calibrating bovine serum albumin or other pure protein solutions for use as standards in other methods. Absorbance assays 2PrinciplevProteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the abso

5、rbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.3vEquipmentvIn addition to standard liquid handling supplie

6、s a spectrophotometer with UV lamp and quartz cuvette are required.4vAnalysisvUnknown proteins or protein mixtures. Use the following formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm. vConcentration (mg/ml) = Absorbance at 280 nm divided by path length (c

7、m.) vPure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used. vconcentration = Absorbance at 280 nm divided by absorbance coefficient vTo convert units, use these relation

8、ships: vMg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight vUnknowns with possible nucleic acid contamination. Use the following formula to estimate protein concentration: vConcentration (mg/ml) = (1.55 x A280) (0.76 x A260) 5vCommentsvCold solutions can fog up th

9、e cuvette, while warm solutions can release bubbles and interfere with the readings. For concentrated solutions (absorbance greater than 2) simply dilute the solution. vAbsorbance coefficients of some common protein standards: vBovine serum albumin (BSA): 63 vBovine, human, or rabbit IgG: 138 vChick

10、en ovalbumin: 70 vReferencesvLayne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 3: 447-455. 1957. vStoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990. 6vAbsorbance Assay (205 nm)vConsiderations for use vSee considerations

11、listed under the absorbance assay at 280 nm. This method is just as convenient as for absorbance at 280 nm. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is rig

12、ht on the shoulder of the protein peak.7vPrinciplevSee the discussion for the 280 nm absorbance assay.vProcedurevInclude 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for the 205 nm assay because losses are proportionately higher in di

13、lute solutions.vWarm up the UV lamp (about 15 min.) vAdjust wavelength to 205 nm vCalibrate to zero absorbance with buffer solution only vMeasure absorbance of the protein solution vAnalysisvProtein concentration (mg/ml) = 31(Absorbance at 205 nm).非离子表示活性剂聚氧乙烯十二烷醇（非离子表示活性剂聚氧乙烯十二烷醇（Brij-35），）， 8vComm

14、entsvCold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere with the readings. Solutions must be much more dilute than for the A280 assay. Proteins absorb much more strongly at 205 nm, and there is supposedly less variability from protein to protein. In additio

15、n to the need for an accurate wavelength setting, stray light can be a major problem. To avoid these problems, use a 10 microgram/microliter solution of bovine serum albumin as a standard. With buffer blank as zero absorbance, determine the concentration of an unknown (concentration between 0 and 10

16、 micrograms/microliter) by interpolation. This is acceptable because of the linear relationship of absorbance and concentration in the 0 to 10 microgram/microliter range. 9vThe problem of an accurate wavelength setting can be avoided by determining absorbance at 210 nm (extinction coefficients range

17、 from 20 to 24). However there is less sensitivity and more variation with buffer conditions. vReferencesvScopes, RK. Analytical Biochemistry 59: 277. 1974. vStoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990. 10vDetermination of the Extinction Coefficient for a Protein o

18、f Unknown ConcentrationvConsiderations for usevThe concentration can be determined for a solution of a pure protein with unknown extinction coefficient.11vEquipmentvIn addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.vProcedurevDilute the

19、 solution about 30 fold for the reading at 205 nm and include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces.12vAnalysisvUse the following formula to determine the extinction coefficient at 205 nm: vE(205 nm) = 27 + 120 x (A280 divided by A205) vThe readi

20、ng at 205 nm must be multiplied by the dilution factor before using the formula. vNext, determine protein concentration: vProtein concentration (M) = A205 divided by E(205 nm) vYou can now determine the extinction coefficient for 280 nm: vE(280 nm) = concentration (M) divided by A280 13vCommentsvAn

21、abnormal phenylalanine content will throw off the result considerably. The accuracy of the technique depends on an average amino acid composition.vReferencesvScopes, RK. Analytical Biochemistry 59: 277. 1974. vStoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990. 14Hartree-

22、Lowry and Modified Lowry Protein AssaysvConsiderations for usevThe Lowry assay (1951) is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on. The bicinchoni

23、nic acid (BCA) assay is based on the same princple and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method is outdated. However, the modified Lowry is done entirely at room temperature. The Hartree version of the Lowry assay, a more recent modifica

24、tion that uses fewer reagents, improves the sensitivity with some proteins, is less likely to be incompatible with some salt solutions, provides a more linear response, and is less likely to become saturated. The Hartree-Lowry assay will be described first.Colorimetric assays: 15vPrinciplevUnder alk

25、aline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungst

26、en blue. 16vEquipmentvIn addition to standard liquid handling supplies a spectrophotometer with infrared lamp and filter is required. Glass or polystyrene (cheap) cuvettes may be used. 17vProcedure - Hartree-Lowry assayvReagentsvReagent A consists of 2 gm sodium potassium tartrate x 4 H20, 100 gm so

27、dium carbonate, 500 ml 1N NaOH, H20 to one liter (that is, 7mM Na-K tartrate, 0.81M sodium carbonate, 0.5N NaOH final concentration). Keeps 2 to 3 months.vReagent B consists of 2 gm 2 gm sodium potassium tartrate x 4 H20, 1 gm copper sulfate (CuSO4 x 5H20), 90 ml H20, 10 ml 1N NaOH (final concentrat

28、ions 70 mM Na-K tartrate, 40 mM copper sulfate). Keeps 2 to 3 months.vReagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.18vAssayvPrepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing the unknowns, to give concentrations of 30 to 1

29、50 micrograms/ml (0.03 to 0.15 mg/ml).vAdd 1.0 ml each dilution of standard, protein-containing unknown, or buffer (for the reference) to 0.90 ml reagent A in separate test tubes and mix.vIncubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature.vAdd 0.1 ml reagent B to each tu

30、be, mix, incubate 10 min at room temperature.vRapidly add 3 ml reagent C to each tube, mix, incubate 10 min in the 50 degree bath, and cool to room temperature. Final assay volume is 5 ml.vMeasure absorbance at 650 nm in 1 cm cuvettes.19vAnalysisvPrepare a standard curve of absorbance versus microgr

31、ams protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.20vProcedure - modified Lowry (room temperature)vReagents vDissolve 20 gm sodium carbonate in 260 ml water, 0.4 gm cupri

32、c sulfate (5x hydrated) in 20 ml water, and 0.2 gm sodium potassium tartrate in 20 ml water. Mix all three solutions to prepare the copper reagent. vPrepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS). vPrepare a 1 M solution of NaOH (4 gm/100 ml). vFor the 2x Lowry concent

33、rate mix 3 parts copper reagent with 1 part SDS and 1 part NaOH. Solution is stable for 2-3 weeks. Warm the solution to 37 degrees C if a white precipitate forms, and discard if there is a black precipitate. Better, keep the three stock solutions, and mix just before use. vPrepare 0.2 N Folin reagen

34、t by mixing 10 ml 2 N Folin reagent with 90 ml water. Kept in an amber bottle, the dilution is stable for several months. 21vAssay vDilute samples to an estimated 0.025-0.25 mg/ml with buffer. If the concentration cant be estimated it is advisable to prepare a range of 2-3 dilutions spanning an orde

35、r of magnitude. Prepare 400 microliters each dilution. Duplicate or triplicate samples are recommended. vPrepare a reference of 400 microliters buffer. Prepare standards from 0.25 mg/ml bovine serum albumin by adding 40-400 microliters to 13 x 100 mm tubes + buffer to bring volume to 400 microliters

36、/tube. vAdd 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min. vAdd 200 microliters 0.2 N Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be accomplished quickly to avoid decomposition of the reagent before it reacts with p

37、rotein. Incubate for 30 min. more at room temperature. vUse glass or polystyrene cuvettes to read the absorbances at 750 nm. If the absorbances are too high, they may be read at 500 nn. 22vComments vRecording of absorbances need only be done within 10 min. of each other for this modified procedure,

38、whereas the original Lowry required precise timing of readings due to color instability. This modification is less sensitive to interfering agents and is more sensitive to protein than the original. As with most assays, the Lowry can be scaled up for larger cuvette sizes, however more protein is con

39、sumed. Proteins with an abnormally high or low percentage of tyrosine, tryptophan, or cysteine residues will give high or low errors, respectively. ReferencesLowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265. 1951. Oostra, GM, NS Mathewson, and GN Catravas. Anal. Biochem. 89:

40、 31. 1978. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). Hartree, EF. Anal Biochem 48: 422-427 (1972). 23Biuret Protein AssayvConsiderations for usevThe principle of the biuret assay is similar to that of the Lowry, however it involves a single incubation of 20 min

41、. There are very few interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more material. The biuret is a good general protein assay for batches of material f

42、or which yield is not a problem. The Bradford assay is faster and more sensitive.24vPrinciplevUnder alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.vEquipmentvIn addition to standard liquid handling supplies a visible light s

43、pectrophotometer is needed, with maximum transmission in the region of 450 nm. Glass or polystyrene (cheap) cuvettes may be used.25vProcedurevReagentvA formula for biuret reagent is 2.25 gm Sodium potassium tartrate (f.w. 282.22), 0.75 gm Copper sulfate x 5 H2O (f.w. 249.68), 1.25 gm Potassium iodid

44、e (166.0), all dissolved in order in 100 ml 0.2 M NaOH (f.w. 40.0). Bring volume to 250 ml with distilled water (scale volume up or down as needed, of course). Discard if a black precipitate forms. 26vAssayvVolumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.

45、 vWarm up the spectrophotometer 15 min. before use. vPrepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction coefficient. The reported sensitive range, using 9 ml reagent to 1 ml sample, is from 1 to 10 mg protein. The actual sensitive range m

46、ay extend beyond the upper limit. vPrepare a reference tube with 1 ml buffer. vIf possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated. vUse 1 ml sample per assay tube vAdd 9 ml Biuret reagent to each

47、 tube, vortex immediately, and let stand 20 min. vRead at 550 nm. 27vAnalysisvPrepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution fact

48、or, if any.28vCommentsvThe color is stable, but all readings should be taken within 10 min. of each other. As with most assays, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups

49、 will give high or low readings, respectively.vReferencesvGornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949. vLayne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957. vRobinson, HW and CG Hogden. J. Biol. Chem. 135:

50、707. 1940. vSlater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269. vWeichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 194629布拉德福德蛋白检测布拉德福德蛋白检测 v使用注意事项使用注意事项 v布拉德福德蛋白检测法非常迅速，而且和劳里法用几乎等量的蛋白质样品. 此法相当精确，对于超出范围的样品可以在几分钟内重新测试。 此法适合一般分析，特别适用于确定的细胞蛋白质含量以及凝胶电