生化分析检测技术课件.ppt

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1、1,2.1.1 吸光光度法,Absorbance Assay(280 nm)Considerations for useAbsorbance 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 linear

2、.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 fractionation

3、 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 recommended for c

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

5、t 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.,3,EquipmentIn addition to standard liquid handling supplies a spectrophotometer

6、 with UV lamp and quartz cuvette are required.,4,AnalysisUnknown proteins or protein mixtures.Use the following formula to roughly estimate protein concentration.Path length for most spectrometers is 1 cm.Concentration(mg/ml)=Absorbance at 280 nm divided by path length(cm.)Pure protein of known abso

7、rbance 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.concentration=Absorbance at 280 nm divided by absorbance coefficient To convert units,use these relationships:Mg protein/ml=%protein divided by 1

8、0=molarity divided by protein molecular weight Unknowns with possible nucleic acid contamination.Use the following formula to estimate protein concentration:Concentration(mg/ml)=(1.55 x A280)(0.76 x A260),5,CommentsCold solutions can fog up the cuvette,while warm solutions can release bubbles and in

9、terfere with the readings.For concentrated solutions(absorbance greater than 2)simply dilute the solution.Absorbance coefficients of some common protein standards:Bovine serum albumin(BSA):63 Bovine,human,or rabbit IgG:138 Chicken ovalbumin:70 ReferencesLayne,E.Spectrophotometric and Turbidimetric M

10、ethods for Measuring Proteins.Methods in Enzymology 3:447-455.1957.Stoscheck,CM.Quantitation of Protein.Methods in Enzymology 182:50-69.1990.,6,Absorbance Assay(205 nm)Considerations for use See considerations listed under the absorbance assay at 280 nm.This method is just as convenient as for absor

11、bance 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 right on the shoulder of the protein peak.,7,PrincipleSee the discussion for the 280 nm absorbanc

12、e assay.ProcedureInclude 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 dilute solutions.Warm up the UV lamp(about 15 min.)Adjust wavelength to 205 nm Calibrate to zero abs

13、orbance with buffer solution only Measure absorbance of the protein solution AnalysisProtein concentration(mg/ml)=31(Absorbance at 205 nm).,非离子表示活性剂聚氧乙烯十二烷醇（Brij-35），,8,CommentsCold solutions can fog up the cuvette,while warm solutions can release bubbles and interfere with the readings.Solutions mu

14、st 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 addition to the need for an accurate wavelength setting,stray light can be a major problem.To avoid these problems,use a 10 microgram/microl

15、iter 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 micrograms/microliter)by interpolation.This is acceptable because of the linear relationship of absorbance and concentration in the 0 to 1

16、0 microgram/microliter range.,9,The problem of an accurate wavelength setting can be avoided by determining absorbance at 210 nm(extinction coefficients range from 20 to 24).However there is less sensitivity and more variation with buffer conditions.ReferencesScopes,RK.Analytical Biochemistry 59:277

17、.1974.Stoscheck,CM.Quantitation of Protein.Methods in Enzymology 182:50-69.1990.,10,Determination of the Extinction Coefficient for a Protein of Unknown ConcentrationConsiderations for useThe concentration can be determined for a solution of a pure protein with unknown extinction coefficient.,11,Equ

18、ipmentIn addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.ProcedureDilute the 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.,12,Anal

19、ysisUse the following formula to determine the extinction coefficient at 205 nm:E(205 nm)=27+120 x(A280 divided by A205)The reading at 205 nm must be multiplied by the dilution factor before using the formula.Next,determine protein concentration:Protein concentration(M)=A205 divided by E(205 nm)You

20、can now determine the extinction coefficient for 280 nm:E(280 nm)=concentration(M)divided by A280,13,CommentsAn abnormal phenylalanine content will throw off the result considerably.The accuracy of the technique depends on an average amino acid composition.ReferencesScopes,RK.Analytical Biochemistry

21、 59:277.1974.Stoscheck,CM.Quantitation of Protein.Methods in Enzymology 182:50-69.1990.,14,Hartree-Lowry and Modified Lowry Protein Assays,Considerations for useThe Lowry assay(1951)is an often-cited general use protein assay.For some time it was the method of choice for accurate protein determinati

22、on for cell fractions,chromatography fractions,enzyme preparations,and so on.The bicinchoninic 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

23、at room temperature.The Hartree version of the Lowry assay,a more recent modification 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-Low

24、ry assay will be described first.,Colorimetric assays:,15,PrincipleUnder alkaline 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

25、 to produce an unstable product that becomes reduced to molybdenum/tungsten blue.,16,EquipmentIn addition to standard liquid handling supplies a spectrophotometer with infrared lamp and filter is required.Glass or polystyrene(cheap)cuvettes may be used.,17,Procedure-Hartree-Lowry assayReagentsReagen

26、t A consists of 2 gm sodium potassium tartrate x 4 H20,100 gm sodium 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.Reagent B consists of 2 gm 2 gm sodium potassium tartrate x 4 H20,1 gm copper sulfate(CuS

27、O4 x 5H20),90 ml H20,10 ml 1N NaOH(final concentrations 70 mM Na-K tartrate,40 mM copper sulfate).Keeps 2 to 3 months.Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.,18,AssayPrepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing

28、 the unknowns,to give concentrations of 30 to 150 micrograms/ml(0.03 to 0.15 mg/ml).Add 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.Incubate the tubes 10 min in a 50 degrees C bath,then cool to room tempe

29、rature.Add 0.1 ml reagent B to each tube,mix,incubate 10 min at room temperature.Rapidly 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.Measure absorbance at 650 nm in 1 cm cuvettes.,19,AnalysisPrepare a standard curv

30、e 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 factor,if any.,20,Procedure-modified Lowry(room temperature)Reagents Dissolve 20 gm sodium carbonate in 260 ml

31、 water,0.4 gm cupric 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.Prepare 100 ml of a 1%solution(1 gm/100 ml)of sodium dodecyl sulfate(SDS).Prepare a 1 M solution of NaOH(4 gm/100 ml).For the 2x Lowry con

32、centrate 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.Prepare 0.2 N Folin reagent by

33、 mixing 10 ml 2 N Folin reagent with 90 ml water.Kept in an amber bottle,the dilution is stable for several months.,21,Assay Dilute 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 order of mag

34、nitude.Prepare 400 microliters each dilution.Duplicate or triplicate samples are recommended.Prepare 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/tube.Add 400 m

35、icroliters of 2x Lowry concentrate,mix thoroughly,incubate at room temp.10 min.Add 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 protein.Incubate for 30 m

36、in.more at room temperature.Use glass or polystyrene cuvettes to read the absorbances at 750 nm.If the absorbances are too high,they may be read at 500 nn.,22,Comments Recording of absorbances need only be done within 10 min.of each other for this modified procedure,whereas the original Lowry requir

37、ed 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 consumed.Proteins with an abnormally hig

38、h 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.89:31.1978.Stoscheck,CM.Quantitation of Protein.Methods in En

39、zymology 182:50-69(1990).Hartree,EF.Anal Biochem 48:422-427(1972).,23,Biuret Protein Assay,Considerations for useThe principle of the biuret assay is similar to that of the Lowry,however it involves a single incubation of 20 min.There are very few interfering agents(ammonium salts being one such age

40、nt),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 for which yield is not a problem.The Bradford assay is faster and more sensitive.

41、,24,PrincipleUnder alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.EquipmentIn addition to standard liquid handling supplies a visible light spectrophotometer is needed,with maximum transmission in the region of 450 nm.Glass

42、or polystyrene(cheap)cuvettes may be used.,25,ProcedureReagentA 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 iodide(166.0),all dissolved in order in 100 ml 0.2 M NaOH(f.w.40.0).Bring volume to 250 ml with dis

43、tilled water(scale volume up or down as needed,of course).Discard if a black precipitate forms.,26,AssayVolumes sample,reagent can be scaled up/down and/or volume ratios varied,as with any assay.Warm up the spectrophotometer 15 min.before use.Prepare standards from bovine serum albumin,preferably ca

44、librated 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 may extend beyond the upper limit.Prepare a reference tube with 1 ml buffer.If possible,dilute unknowns to an estima

45、ted 1 to 10 mg/ml with buffer;a range of dilutions should be used if the actual concentration cannot be estimated.Use 1 ml sample per assay tube Add 9 ml Biuret reagent to each tube,vortex immediately,and let stand 20 min.Read at 550 nm.,27,AnalysisPrepare a standard curve of absorbance versus micro

46、grams 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.,28,CommentsThe color is stable,but all readings should be taken within 10 min.of each other.As with most assays,the Biuret

47、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 will give high or low readings,respectively.ReferencesGornall,AG,CS Bardawill,and MM David.J.Biol.Chem.177:751.1949.Layne,E.Spectrophotometr

48、ic and Turbidimetric Methods for Measuring Proteins.Methods in Enzymology 10:447-455.1957.Robinson,HW and CG Hogden.J.Biol.Chem.135:707.1940.Slater,RJ(ed.).Experiments in Molecular Biology.Clifton,New Jersey:Humana Press,1986.P.269.Weichselbaum,TE.Am.J.Clin.Pathol.Suppl.10:40.1946,29,布拉德福德蛋白检测,使用注意事

49、项 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泳蛋白的含量分析。,30,Assay materials including color reagent,protein standard,and instruction booklet are available from Bio-Rad Corporation.The method described below is for a 100 l sample volume using 5 ml color re

50、agent.It is sensitive to about 5 to 200 micrograms protein,depending on the dye quality.In assays using 5 ml color reagent prepared in lab,the sensitive range is closer to 5 to 100 g protein.Scale down the volume for the microassay procedure,which uses 1 ml cuvettes.Protocols,including use of microt