Spectrophotometry Handbook - Sigma
Contents
1. 330 to 800 nm Novaspec Plus As Novaspec Ill with stored protein methods easy to read graphical display and temperature control option Ultrospec 10 Battery powered portable cell density meter ooom ooo Key Recommended Suitable Not suitable 21 GE Healthcare spectrophotometer range NanoVue Plus GeneQuant 1300 Novaspec III amp Plus Ultrospec 10 Small and simple UV Good all round UV Visible Small and simple Portable battery Visible spectrophotometer instrument with pre protein measurement operated cell density for measuring small stored methods for DNA meter for bacterial cell volumes between 0 5 and protein analysis culture measuring ODsoo and 5 ul Pre stored methods for DNA and proteins no cuvettes required Just Drop Measure Done For most molecular For research and Ideal for teaching labs For cell culture labs biology and teaching labs and some research labs biochemistry labs Versatile UV Visible Flexible economical Economical solution A simple solution spectro that quickly and solution multivolume perhaps multiple for measuring accurately quantifies perhaps multiple instruments where bacteria cell cultures nucleic acids and protein instruments UV needed UV is not needed avoiding contamination samples preferably for DNA RNA measures Note Novaspec does not within seconds GeneQuant does most do UV so if UVis needed If a PC piloted instrument APplications and if measurin2. In a UV Vis spectrophotometer a monochromator is the optical device that transmits a mechanically selected band of wavelengths of light from a wider range of wavelengths available from the light source From the Greek mono single and chroma color Pathlength Pathlength Is the distance through the sample through which the light has to pass in order to reach the detector Standard cuvettes have a pathlength of 10 mm 1 cm GE Healthcare offers instruments covering pathlengths from 0 2 mm all the way to 100 mm Stray Light Stray light is light that reaches the detector that is of a different wavelength to that the monochromator is selecting This can be caused by the diffraction pattern produced by the monochromator or from light leaks from the outside environment GE Healthcare systems are designed to reduce all possible sources of stray light UV Vis The electromagnetic spectrum is split into a number of different regions by wavelength These include X ray 0 01 10 nm Visible light 380 760 nm and Microwaves 1 10cm GE Healthcare UV Vis spectrophotometers all measure in the UV 10 400 nm visible 380 760 nm and near infra red 750 2250 nm regions covering a range of 190 1100 nm Wavelength Represented by the lower case Greek letter lambda A Wavelength is a measurement of the distance between successive peaks in a waveform L Wavelength Distance gt 27 References 28 J H Lambert 1760 Photomet
3. at a point in the spectrum away from the wavelength of interest and this reading is subtracted from the main result This removes the likelihood of any interference Sources of interference may include e Poor drop placement This causes incomplete light transition though the sample If the light path intersects with the end of the sample light is refracted preventing it from reaching the return optics so it could be counted as absorption e Air bubbles in sample As with poor drop placement air drops cause refraction of the light from its normal path which can in turn cause some unabsorbed light not to reach the detector and thus increasing apparent absorption e Particles in sample Particle contamination in a sample causes light scatter preventing unabsorbed light reaching the detector Again this will increase the apparent absorption 18 Which spectrophotometer Some general features and questions to consider when choosing your next instrument are How does your data need to be recordedor How many users will there be and with what stored Consider whether standard PC level of experience Pre programmed software is suitable or whether methods and password control of electronic records and audit your own methods may trails are required be useful What range of sample volumes do you need What level of to measure sensitivity do you need Do you want to measure multiple samples at the same time What applications are
4. is provided for information amp must be subtracted from the Azso nm reading to arrive at the absorbance e For information Not all low volume instruments display the A329 nm subtraction in the calculation Protein applications As with DNA proteins absorb light at a specific wavelength allowing direct measurement using a spectrophotometer The amino acids Tyrosine and Tryptophan have a very specific absorption at 280 nm allowing direct Azg measurement of protein concentration Direct UV measurement at 280 nm has many advantages since the protein solution alone is used without the addition of reagents and it is not modified or inactivated during the process No incubation period is required so measurements are quick and highly reproducible The chemical composition of the protein will affect the absorption the number as well as the type of amino acids will cause variation How much a protein absorbs at 280 nm is dependent on the amount of the amino acids Tyrosine and especially Tryptophan the aromatic ring of Phenylalanine absorbs well at 260 nm but not 280 nm So proteins of similar molecular weight can have quite different absorbances since they can have completely different Tryptophan and Tyrosine content UV absorbance of aromatic side chains is also affected by protein structure Therefore conditions which affect structure such as temperature pH ionic strength or the presence of detergents can affect the ability of aromatic resid
5. life without reducing output called press to read Pulse technology This allows the lamp to go into a low power mode where the lamp is kept warm but is not actually on This can increase the lamp life up to 3 times that of a conventional deuterium lamp The deuterium lamps of the GE Healthcare Ultrospec 8000 and 9000 can be changed by the researcher in the lab Deuterium lamps are paired with tungsten lamps to allow coverage of the full UV and visible spectrum Tungsten Tungsten lamps give stable light in the visible and near infra red areas of the spectrum covering 320 1100 nm Fig 10 GE Healthcare tungsten lamps also employ press to read Pulse technology to extend lamp life and the lamps in the Ultrospec 8000 and 9000 can also be changed by the researcher in the lab 25 _ Deuterium f arc lamp Tungsten halogen lamp Energy gt 200 400 600 800 Anm Fig 10 Both Deuterium and Tungsten lamps are required in order to cover the complete wavelength range from 190 nm to 1100 nm xenon Xenon flash lamps are a high energy light source emitting light across the UV Visible and near IR spectrum They are known as flash lamps as they are not on constantly but flash up to 80 times a second Xenon lamps have a very long life span but don t provide the optical stability of the deuterium tungsten set up in the higher specification systems Xenon lamps must be changed by a service engineer The entry level dual be
6. oil extra virgin virgin etc One section looks at the absorption values for 232 nm and 270 nm this is a measurement of oxidation To be classed as extra virgin olive oil A232 has to be lt 2 5 and A27 lt 0 22 For virgin olive oil these values are slightly higher at 2 6 and 0 25 A spectrophotometer is essential for the grading of olive oil in line with European regulations The absorbance spectrum for the molecule to be measured may vary from very narrow e g lt 1 nm to broad e g gt 3 nm If extreme accuracy is required for example in the quality control of small molecules it may therefore be necessary to alter the bandwidth of the instrument accordingly Instruments such as the Ultrospec 9000 have variable bandwidth to enable more precise measurements in method development and small molecule measurements For more information see Bandwidth in glossary 16 Spectrophotometry hints and tips Choosing a cuvette Beam heights The height at which the light path of the spectrophotometer hits the cuvette is known as the beam or Z height Most common beam heights are 8 5 mm 15 mm and 20 mm It is important to use cuvettes which are designed for the correct beam height firstly to ensure they are optically clear at the point the light hits the surface and secondly because the stated pathlength will be at this exact point in the cuvette and may differ at other heights on the cell Some manufacturers offer packing pieces to allow th
7. GE Healthcare Life Sciences Spectrophotometry Handbook Handbooks from GE Healthcare Life Sciences Imaging Principles and Methods 29 0203 01 GST Gene Fusion System Handbook 18 1157 58 Affinity Chromatography Principles and Methods 18 1022 29 Antibody Purification Handbook 18 103 7 46 lon Exchange Chromatography and Chromatofocusing Principles and Methods 11 0004 21 Cell Separation Media Methodology and Applications 18 1115 69 Purifying Challenging Proteins Principles and Methods 28 9095 31 Isolation of mononuclear cells Methodology and Applications 18 1152 69 High throughput Process Development with PreDictor Plates Principles and Methods 28 9403 58 Protein Sample Preparation Handbook 28 9887 41 aa e ii ete Di cE pacat _ Cel Care AKTA Laboratory scale Chromatography Systems Instrument Management Handbook 29 0108 31 Gel Filtration Principles and Methods 18 1022 18 Recombinant Protein Purification Handbook Principles and Methods 18 1142 75 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11 0012 69 2 D Electrophoresis using immobilized pH gradients Principles and Methods 80 6429 60 Microcarrier Cell Culture Principles and Methods 18 1140 62 Nucleic Acid Sample Preparation for Downstream Analyses Principles and Methods 28 9624 00 Western Blotting Principles and Methods 28 9998 97 Strategies for Prot
8. am Ultrospec 7000 instrument has a xenon lamp LED Used for single wavelength applications such as cell culture measurement in the Ultrospec 10 LEDs are stable low cost and offer a long life Methods of measurement Single wavelength A simple method which allows measurement of transmittance t or absorbance of a substance at a single fixed wavelength E g 260 nm for DNA Wavelength scan Measurements for a range of wavelengths are taken allowing the system to plot absorbance against wavelength and produce a graph Particularly useful for analysing an unknown sample The start and finish wavelengths can be set to target the scan to a region or the available spectrum Kinetics A single wavelength measurement is repeated at set intervals for a set number of times The absorption is then plotted against time This can be used to measure reaction progress or for enzyme activity studies when combined with a temperature controlled cell holder 26 Quantitative analysis A single wavelength measurement of a number of Known standards can be taken from which the instrument creates a standard curve to which future measurements of unknown samples can be compared to establish their concentration Cell Density Light scatter from bacterial cell cultures can be used as an indication of cell concentration and when plotted over time can be used to see if the culture is ready to be harvested Measurements are taken at 600 nm Monochromator
9. dy To Go ambient temperature Stable products e E E Protein Sample Preparation Mag Sepharose MiniTrap MultiTrap SpinTrap GraviTrap for simple protein sample prep including for recombinant proteins and antibodies Chromatography media and protein purification TALONS HisTrap GSTrap HiTrap Tricorn k MonoBeads complete range of media and Ti columns for protein purification v Amersham DNA and Protein Labeling Blotting and Detection CyDye dyes HyPer5 microarray dyes for array CGH and gene expression ECL Labeling and Detection range ECL Gel Box Rainbow Markers Hybond Membranes Hyperfilm Whatman filtration and sample prep range FTA e Core Whatman sterile syringe filters Puradisc FP30 for buffer and sample filtration Whatman GD x for filtration of particulate loaded samples 29 For local office contact information visit www gelifesciences com contact www gelifesciences com spectros GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company Cy CyDye Datrys e Core ECL ExoProStar FTA GeneQuant GenomiPhi GFX GraviTrap GSTrap HisTrap HiTrap Hybond Hyperfilm illustra Mag Sepharose MiniTrap MonoBeads MultiTrap NanoVue Novaspec Pharmalyte Rainbo
10. e sample preparation phase and choose a non absorbing buffer or one which will not interfere with the sample analysis Studies have found that some detergents have more of an effect on direct UV results than others Using detergents such at Brij 35 CHAPS and Tween 20 may give a better results than Igepal or Triton x 100 but this will vary greatly on the concentration of detergent present in the sample 17 Low volume spectrophotometers Choice of volume For low volume spectrophotometers the pathlength is generally much smaller than for cuvette based instruments e g 0 1 mm to 1 mm and may rely on the sample itself to form the cuvette Therefore contact with both the upper and lower sample plates heads is critical For best results when using volumes below 2 ul with NanoVue select the 0 2 mm pathlength For volumes of 2 ul or above select the 0 5 mm pathlength Background correction If your low volume instrument has the possibility to apply background correction as with Nanovue it should always always be switched on because it can e Correct for drop misplacement e Correct for sample background value of sample eg DNA should have zero absorbance at 320 nm Any reading at 320 nm can indicate a contaminant or faulty drop placement As a rough guide the expected 320 nm value should be approximately Yo of the 260 nm reading Background correction is very important when using low volume spectrophotometers A reading is taken
11. e use of cuvettes designed for lower beam heights on their systems Choice of cuvettes The first decision when choosing a cuvette type is whether you wish to use disposable cells or cuvettes designed to be cleaned and reused Disposable cuvettes remove the need for a cleaning process within the laboratory removing the chance of carry over but they add an on going cost to running the analyzer have higher native absorbencies and may not be produced to the same precise standards as reusable items Reusable cuvettes are much more expensive than their disposable counterparts and when using a dual beam spectrophotometer the cuvettes need to be matched to ensure accurate blank readings The second decision is the choice of cuvette material Cuvettes are generally split into use for visible only and UV vis Normal plastics and glass naturally absorb UV light so a glass cuvette would not be suitable for DNA analyses but would be fine for cell culture measurements UV cuvettes are more expensive than their visible alternatives as they need to be made from specialist plastics or quartz glass Buffer Compatibility There are many commonly used processes and methods in use for biological sample preparation and purification and these often require the use or a detergent or buffer Many buffers and detergents absorb light themselves interfering with the spectrophotometric analysis of the sample It is important to take this into account when designing th
12. ein Purification Handbook 28 9833 31 Contents Spectrophotometry basics 3 What is spectrophotometry 3 Definition 3 Lambert s Law 4 Beer s Law 4 Nucleic acid applications 6 Direct UV measurement 7 A260 A280 Ratio 7 A260 A230 Ratio 7 A320 Background correction 7 Nucleic acid measurements with low volume instruments 8 Protein applications 9 Protein concentration calculations 10 Christian and Warburg 10 Colorimetric methods 10 BCA 11 Biuret 11 Bradford 11 Lowry 11 2 D Quant Kit 11 Choosing your protein assay 12 Measuring proteins in low volume instruments 14 Other applications 15 Fluorescent dyes is Cell culture 15 Other molecules 16 Continued overleaf Spectrophotometry hints and tips Choosing a cuvette Beam heights Choice of cuvettes Buffer Compatibility Low volume spectrophotometers Choice of volume Background correction Which spectrophotometer Spectrophotometer selection guide GE Healthcare spectrophotometer range Datrys PC control software Accessories overview Glossary Absorption Bandwidth Beam technology Light source comparison Methods of measurement Monochromator Pathlength Stray Light UV Vis Wavelength References Related products 17 17 17 17 I7 18 18 18 19 21 22 22 23 24 24 24 25 25 26 27 27 27 27 27 28 29 Spectrophotometry basics What is spectrophotometry Spectrophotometry is a scientific method based on the absorption of light by a substance a
13. from the absorption readings To compensate for this a selection of ratios and background corrections have been developed to help eliminate false readings 0 9 X 300 Y 0 063 0 8 0 7 0 6 0 5 Absorbance 0 4 0 3 0 2 0 1 0 0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 Wavelength nm Fig 4 A typical wavelength scan for a pure DNA sample There is a wide absorbance peak around 260 nm preceded by a dip at 230 nm Therefore to measure the DNA absorption the 260 nm DNA peak must be distinguishable from the 230 nm reading If the readings at 230 nm are too similar to those at 260 nm DNA cannot be measured accurately Higher 230 nm readings can indicate contaminants in the sample There should also be a rapid tail off from 260 nm down to 320 nm For this reason 320 nm is often used to measure background see background correction Direct UV measurement A260 A280 Ratio The most common purity check for DNA and RNA is the Az6o Azao ratio Any protein contamination will have maximum absorption at 280 nm Measurements are taken at both 260 nm and 280 nm and compared to give a ratio For DNA the result of dividing the 260 nm absorption by the 280 nm needs to be greater or equal to 1 8 to indicate a good level of purity in the sample For RNA samples this reading should be 2 0 or above Results lower than this are indicative of impurities in the sample A260 A230 Ratio A
14. g DNA or is needed Datrys Life capillary cell can go RNA prefer GeneQuant Science software down to 3 ul volumes is available Datrys PC control software An external PC with appropriate software allows the ultimate in flexibility to control and manipulate spectrophotometry data Whether looking for small differences in multiple spectral overlays or carrying out post run manipulations on large numbers of samples Datrys software from GE Healthcare has the flexibility to work in the way you want Available in four options Datrys Lite Datrys Standard Datrys Life Science and Datrys CFR and compatible with Windows XP Windows Vista and Windows 7 and 8 operating systems Data export options include Microsoft Word and Excel plus Adobe PDF formats Datrys software allows PC control of Ultrospec 7000 8000 and 9000 and also PC control a number of other GE Healthcare spectrophotometers including the NanoVue and GeneQuant 22 Ultrospec 2100 UV Visible 3 nm bandwidth instrument wavelength range 190 to 900 nm with NEW Ultrospec 7000 Entry level dual beam instrument with a xenon light source Touchscreen USB data NEW Ultrospec 8000 Dual beam Deuterium tungsten light sources Touchscreen USB data storage and PC control NEW Ultrospec 9000 Dual beam Deuterium tungsten light sources Touchscreen USB data storage and PC an 8 position cell changer and options for temperature control sto
15. g pathlength and compare the results directly with each other GE Healthcare offers a variety of instruments and accessories which allow measurement of pathlengths from 10 cm down to 0 2 mm Ls 1 2 Absorbance ODO LO O 0 3 0 0 0 0 0 2 0 4 0 6 0 8 1 0 Path Length cm Fig 2 Beer s Law The absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium Short pathlength instruments are used when the sample is of limited volume scarce and maybe requiring recovery or is very concentrated e g gt 50 ug DNA ml and the user wishes to avoid the need for dilution Many samples would traditionally require dilution for two reasons 1 So that there is enough volume to fill a 10 mm pathlength cuvette 2 To lower the sample concentration enough to allow accurate measurement by the spectrophotometer Dilution introduces an aspect of human error and can also prevent the use of that sample in downstream applications To measure concentrated samples using a 10 mm pathlength would require a very powerful light source to give transmittance that is high enough to be detected reliably A shorter pathlength reduces the absorbance increasing the transmittance hence reducing the incident light required to achieve a reliable result This removes the need to dilute the sample or to have a larger more powerful or more expensive instrument When using short pathlength
16. ications Fluorescent dyes Many spectrophotometers come with pre programmed methods for analysis of fluorescent dyes for DNA or protein quantification DNA yield can be measured at 260 nm while the incorporation of fluorescein Cy 3 Cy5 and other dyes are measured at their respective absorption peaks This method is also useful for measuring the yields and brightness of fluorescently labeled in situ hybridization probes Cell culture Specialist cell culture spectrophotometers are available for measuring cell density which can be used to measure the growth curve of a cultured population to allow harvesting of cells typically bacteria at the optimum point The basic method of measurement is to analyze light scatter at 600 nm the greater the number of cells the larger the amount of light scattered as it passes through the cuvette and if more light is scattered less light reaches the detector Not all spectrophotometers can measure bacterial cell culture at 600 nm the instruments need to be designed to restrict the amount of scattered light that can reach the detectors More advanced spectrophotometers also offer background correction at 800 nm and algorithms to give more consistent results Cell culture measurements are not quantitative but are used to gauge the current point of the cell culture process and identify the peak concentration for most effective harvesting As the bacterial population approaches its maximum the growth rate wi
17. just passes through the sample holder straight to the detector An initial reference is required to standardize the instrument before analysis can begin Single beam instruments are generally very simple and economical to purchase Examples of such instruments are Novaspec Ill amp Plus Split Beam The light from the source is split into two paths with approximately 30 of the energy being diverted from the main path into a feedback detector 70 is then passed through a monochromator through the sample compartment to a detector The feedback detector is used to correct variations in the energy emitted by the lamp A reference sample is required at the start of each run to correct for cuvette and solvent absorption Dual Beam Sometimes known as double beam technology The light is split into two paths each of which passes through a cell holder onto its own detector One cell holder is for the sample and the second is for a reference The reference should contain the same cuvette type and solvent as the sample The absorption of the reference path is subtracted from that of the sample path Every measurement has its own reference giving more reliable results GE Healthcare Ultrospec 7000 8000 and 9000 are all Dual beam instruments Light source Deuterium Deuterum arc lamps provide powerful and stable light in the UV region 190 370 nm Fig 10 In higher specification spectrophotometers a special design of deuterium lamp is used to maximize
18. ll slow and plateau before beginning to drop off as the cells begin to die due to lack of nutrients and increase in toxic waste products from the growth process 15 The numerical results from different spectrophometer designs are not directly comparable but the same pattern of increase level and decrease will be seen on all instruments Instrument 1 Instrument 2 lt lt All scattered light hits the detector Not all scattered light hits detector therefore a higher absorbance reading is obtained Fig 8 Different spectrophotometers will give different readings when such readings are related to light scatter e g for bacterial culture measurement Depending on the exact set up of an individual spectrophotometer the light scatter and thus the absorbance reading are likely to be different as seen here Features of the instrument design will affect the way in which scattered light can reach the detector and hence the numerical results e g The distance from the sample holder to the detector lens Other molecules As well as biomolecules such as Proteins and DNA there are a wide range of light absorbing substances which it is important to quantify Spectrophotometers are used in a wide range of industries from waste water analysis to pharmaceutical quality control and food analysis One such example is the grading of olive oil The European regulations ECC 2568 91 and ECC 2472 97 set out the characteristics for grading of olive
19. n increase in absorbance at 230 nm can also indicate contamination which may in turn affect the 260 nm reading for DNA and RNA A number of substances absorb at 230 nm as this is the region of absorbance of peptide bonds and aromatic side chains Several buffer components exhibit strong absorption at 260 nm and therefore can alter the results of photometric quantification One example of such a component is EDTA in concentrations above 10 mM Contaminants in a sample such as proteins phenol or urea can result in absorption at 230 nm Phenol contamination also increases a sample s absorption at 280 nm and therefore can be identified through a lower Az6o Azgo ratio AN Az6o Az30 ratio of 2 or above is indicative of a pure sample A320 Background correction Background correction is a process whereby the absorption at a point on the spectrum unrelated to the sample being analyzed is also measured and the reading subtracted from the peaks Absorption at 320 nm may be due to light scatter caused by particles or to a precipitate in the sample Dirty or damaged cuvettes can cause absorption at 320 nm Contaminations with chaotropic salts such as Nal can also lead to increased light scatter Measuring and correcting for the reading at 320 nm therefore removes any interference from light scatter from the cuvette or in cases where a blanking plate is used to target the light beam though the sample Background correction is particularly useful when
20. n protein variation effects and improve assay sensitivity Working Range Kits 1000 to 15 000 ug ml Advantages Disadvantages 13 Wide working range Single reagent single incubation performed at RT easy to use Relatively low sensitivity Relatively large amounts of protein sample needed for accurate analysis Amino buffer e g Tris used in pH range 8 10 can interfere with reaction Sensitive to interference by strong reducing agents Measuring proteins in a low volume instruments NanoVue Plus Spectrophotometer can be used to determine the concentration of protein samples by a variety of methods including Bradford BCA Lowry Biuret and direct UV methods with a choice of line fit and the ability to run up to 27 standards including replicates These calibration curves can be viewed on the graphical display printed or stored as a method for future use gt 595 2 0 1 5 1 0 1 0 0 0 0 500 1000 1500 2000 BSA ug ml Fig 6 Bradford protein assay curve generated from samples measured on NanoVue Plus n 3 replicates Mean 1SD The sample volume was 4 ul 12 0 2500 5000 7500 10 000 12 500 15 000 BSA ug ml Fig 7 Protein concentration curve generated from 2 ul BSA samples measured at 280 nm using NanoVue Plus n 3 replicates Mean 1SD More information can be found in Use of NanoVue spectrophotometer to measure protein concentrations Application Note 28 9468 37 14 Other appl
21. nd takes advantage of the two laws of light absorption Definition specttro pho tom e ter spektrofo tamitar Noun An apparatus for measuring intensity of light in a part of the spectrum as transmitted or emitted by particular substances Visible Light Infrared Ultraviolet 1mm Sum 1400nm 750 nm 700 nm 650 nm 600 nm 550 nm 500 nm 450 nm 400 nm 320 nm280 nm 200 nm Fig 1 The electromagnetic spectrum GE Healthcare Life Sciences offers a range of spectrophotometers which operate in the UV visible and near infrared section of the electromagnetic spectrum Lambert s Law 1 The proportion of light absorbed by a medium is independent of the intensity of incident light A sample which absorbs 75 25 transmittance of the light will always absorb 75 of the light no matter the strength of the light source Lambert s law is expressed as l l T Where I Intensity of transmitted light Intensity of the incident light T Transmittance This allows different soectrophotometers with different light sources to produce comparable absorption readings independent of the power of the light source Beer s Law 2 The absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium In Spectrophotometry the thickness of the medium is called the pathlength In normal cuvette based instruments the pathlength is 10 mm Beer s law allows us to measure samples of differin
22. output to the light that has passed through the sample Bandwidth Bandwidth describes the property of the light emitted from the monochromator When the light is analyzed the peak may be at its maximum at the specified wavelength but light is also emitted to a lesser extent at the wavelengths either side of the one selected The Spectral Bandwidth is a measurement of the width of a peak at half light intensity Fig 9 Half Height Intensity Spe Band Wavelength Fig 9 The Bandwidth is a measurement of the width of a peak at half light intensity To obtain maximum sensitivity the bandwidth of the instrument would cover the entire bandwidth of the substance being measured When reducing bandwidth the amount of available light for absorption is also reduced so more sensitive detectors are needed to obtain the same working range Increasing bandwidth reduces resolution and measurement at too broad a bandwidth could also possibly merge two absorption peaks into one larger one giving an artificially high reading Low bandwidth instruments are very important in highly regulated environments such as pharmaceutical and Pharmacopoeia laboratories where resolution of 1 nm is often required whereas a broader bandwidth is helpful for sensitive DNA quantification where absorption is strong and only one broad 5 nm peak is being measured 24 Beam technology Single beam Single beam refers to the fact that the light
23. r structure in the proteins will influence the absorption Detection can be influenced by nucleic acids and other UV absorbing contaminants and by light scattering from particles in the sample 1 to 1500 ug ml Dye reagent stable for 1 hour More stable than Azgo 2 5 25 micro assay Absorbance spectra of 2 species partially overlap bound v unbound Stains labware on contact Susceptible to interference by high detergent concentrations in solution Precipitates can form High protein to protein signal variability 1 to 1500 ug ml Sensitive over wide working range Commonly referenced procedure for protein determination Reaches a stable end point Performed at room temperature RT Susceptible to interference from wide range of substances Reagent time consuming to prepare Reagent must be prepared fresh each time Photosensitive assay Amount of color varies with different proteins Precipitate can form in the presence of detergents Interference from carbohydrates and some buffers Micro BCA Working Range Kits 1 to 40 ug ml standard BCA range 125 to 2000 ug ml Advantages Disadvantages Very sensitive Not as susceptible to interference from common buffer substances eg many detergents Low protein to protein variability Concentrated protein samples need diluting Sensitive to interference by strong reducing agents Requires longer incubation e g 1 hr at higher temperatures e g 37 C 60 C to minimize protei
24. rage and PC control option 2 nm bandwidth Pharmacopeia compatible control Pharmacopeia 1 nm bandwidth compatible Advanced method development variable bandwidth 0 5 1 2 and 4 nm For research labs core facilities For some pharma some research labs core facilities For pharma some research labs For pharma high specification labs Diverse applications UV Visible Flexibility for measuring multiple samples including for temperature controlled enzyme kinetics Multiple applications dual beam instrument Method development extreme accuracy Pharmacopeia compatibility Multiple applications extreme accuracy Pharmacopeia compatibility Accessories overview Different accessories are available to expand the capabilities of GE Healthcare spectrophotometers These include multiple cell changers temperature regulated cells sipper system for automated sample aspiration test tube holders etc A range of cuvettes are available from capillaries gt 3 ul working volume ultra microcells 7 ul volume to standard quartz cells up to 2500 ul The full range of accessories can be found at www gelifesciences com spectros 23 Glossary Absorption Absorption is the amount of light that a substance takes in and does not allow to pass through it Soectrophotometers actually measure transmission the amount of light that passes through a sample but this is converted into absorption by comparing the bulb
25. ria sive de mensura et gradibus luminis colorum et umbrae Photometry or On the measure and gradations of light colors and shade Augsburg Augusta Vindelicorum Germany Eberhardt Klett Beer 1852 Bestimmung der Absorption des rothen Lichts in farbigen Flussigkeiten Determination of the absorption of red light in colored liquids Annalen der Physik und Chemie vol 86 pp 78 88 Warburg O and Christian W 1942 Isolierung und Kristallisation des Garungsferments Enolase Biochem Z 310 384 421 Smith PK et al 1985 Measurement of protein using bicinchoninic acid Anal Biochem 150 1 76 85 3843705 Layne E 1957 Spectrophotometric and Turbidimetric Methods for Measuring Proteins Methods in Enzymology 10 447 455 Bradford M M 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding Anal Biochem 72 248 254 Lowry O H Rosebrough J J Farr A L and Randall R J 1951 Protein measurement with the Folin phenol reagent J Biol Chem 193 265 275 Related products GE Healthcare s spectrophotometer range and life sciences consumables are now available from your preferred supplier Contact your local GE Healthcare representative for more details illustra Nucleic Acid Preparation Purification plasmid DNA genomic DNA RNA sequencing clean up GenomiPhi amplification ExoProStar GFX PCR clean up Rea
26. routinely run in Do you need your laboratory Could this change in the near future Pharmacopoeia compatibility Consideration of system specification is a fine balancing act between bandwidth absorbance range system noise and cost Ideally this would be a narrow bandwidth instrument with a wide absorbance range low noise and low cost However reducing the bandwidth reduces the available light in turn reducing the absorbance range available To overcome this one could use a more powerful light source and or more sensitive detectors which will increase system noise increasing the lower limit of detection Higher specification electrical systems and detectors will help reduce noise but generally at a much higher cost than most laboratories can afford 19 GE Healthcare has a range of spectrophotometers for a broad range of scientific needs Ultrospec 10 for cells Ultrospec 9000 Novaspec variable band width basic vis absorption advanced applications and transmission kinetics method development Ultrospec 8000 GeneQuant Spectrophotometer Pharmacopoeia compatible dual beam analysis flexible for DNA RNA range protein cultures Ultrospec 7000 economical dual beam functionality Ultrospec 2100 lab standard DNA RNA protein kinetics up to 8 samples NanoVue micro volume drop measure done simplicity 20 Spectrophotometer selection guide DNA RNA quantitation PCR sequencing hybridiza
27. s less than 10 mm results are generally normalized to that of a 10 mm pathlength e g In the case of a 0 2 mm pathlength the absorbance results are multiplied by 50 However at the same time any error from the system of absorption by the cuvette is also multiplied by 50 increasing the possible effect on the result In basic terms Absorbance Concentration x Pathlength Light Source Adjustable Monochromator Aperture Photoresistor Amplifier Spectrum af Display of light ew Light is Light is absorbed detected Measure is displayed Fig 3 General schematic of a spectrophotometer Nucleic acid applications Spectrophotometry can be used to estimate DNA or RNA concentration and to analyze the purity of the preparation Typical wavelengths for measurement are 260 nm and 280 nm In addition measurements at 230 nm and 320 nm can provide further information Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm For pure samples it is well documented that for a pathlength of 10 mm an absorption of 1A unit is equal to a concentration of 50 ug ml DNA and 40 ug ml for RNA For oligonucleotides the concentration is around 33 ug ml but this may vary with length and base sequence So for DNA Concentration ug ml Absz60 x 50 These values are known as conversion factors A number of other substances which also absorb light at 260 nm could interfere with DNA values artificially increasing the result calculated
28. s an absorbance peak maximum at 562 nm Most commercially available kits state that this method is designed to quantify 125 to 2000 ug ml Biuret 5 Like BCA the Biuret method takes advantage of the reaction between cupric ions and peptide bonds in alkali solution In this case no additional development step is used Absorption Is measured at 546 nm Most commercially available kits state that this method is designed for the quantification of 1000 to 15 000 ug ml Bradford 6 This method uses a dye Coomassie Brilliant Blue which binds to the protein The protein solution shows an increase in absorbance at 595 nm which is proportional to the amount of bound dye Bradford is very resistant to interference making it a very common method Most commercially available kits state that the useful range of this method is 1 to 1500 ug ml Lowry 7 Lowry takes advantage of the reaction of Folin Ciocalteu s phenol reagent with tyrosyl residues of an unknown protein The absorption at 750 nm can then be compared to a standard curve of a known protein normally BSA Most commercially available kits state that this method is useful to quantify 1 to 1500 ug ml 2 D Quant Kit The 2 D Quant Kit 80 6483 56 is designed to determine protein concentration in samples to be analyzed by high resolution electrophoresis techniques such as 2 D electrophoresis SDS PAGE or IEF Many reagents used in preparing such samples including detergents reductants chao
29. tion Primer quantitation and design Protein determination Bradford Biuret BCA etc Cell culture ODsoo Enzyme activity kinetics at 340 nm Fluorescent probe quantitation cDNA probes for microarrays PCR probes Pharma method development Main features Ultrospec 9000 High performance dual beam variable bandwidth UV Visible spectrophotometer Wavelength range 190 1100 nm European Pharmacopeia compatible Optional Life Science or CFR Datrys software upgrade available Ultrospec 8000 High performance dual beam 1 nm bandwidth UV Visible spectrophotometer Wavelength range 190 1100 nm European Pharmacopeia compatible Optional Life Science or CFR Datrys software upgrade available Ultrospec 7000 High performance dual beam 2 nm bandwidth UV Visible spectrophotometer Wavelength range 190 1100 nm Optional Life Science or CFR Datrys software upgrade available Ultrospec 2100 pro 3 nm bandwidth instrument wavelength range 190 to 900 nm with 8 position cell changer and options for temperature control NanoVue Plus Novel sample plate No cuvettes needed PC control now available Low volume measurements 0 5 to 5 ul GeneQuant 1300 As GeneQuant 100 with built in enzyme kinetics and CyDye applications plus Bluetooth data output options GeneQuant 100 5 nm bandwidth instrument with built in applications for nucleic acids proteins and cell density Novaspec III 7 nm bandwidth visible spectrophotometer
30. tropes and carrier ampholytes are incompatible with other protein assays The procedure quantitatively precipitates proteins leaving interfering substances in solution The assay is based on the specific binding of copper ions to protein Precipitated proteins are resuspended in a copper containing solution and unbound copper is measured with a colorimetric agent The color density is inversely related to the protein concentration The assay is linear in the range of 0 50 ug protein The procedure is compatible with 2 SDS 1 DTT 8 M urea 2 M thiourea 4 CHAPS 2 Pharmalyte and 2 IPG Buffer Absorbance is read at 480 nm See the following pages for a Summary you can use for choosing your protein assay method Please refer to the kit manufacturer s product data sheets for the exact ranges for your specific kit 11 Choosing your protein assay Working Range Advantages Disadvantages Working Range kits Advantages Disadvantages Working Range Kits Advantages Disadvantages 100 ug ml to approx 100 000 ug ml This range is valid for low volume spectrophotometers such as NanoVue linearity in NanoVue 100 100 000 ug ml 100 ug ml based on 2 SD 20 replicates gt 10000 ug ml 1 20 replicates CV For a standard instrument working range is 0 05 to 2000 ug ml Simple direct UV measure minimizes need for dilution Suited to identifying protein on column fractions Wide working range Higher orde
31. ues to absorb light at 280 nm and change the value of the protein s extinction coefficient As with nucleic acids each protein has its own conversion factor The common standard protein bovine serum albumin BSA has a factor of 1 551 Concentration ug ml AbS2so x Factor The Azeo Azgo ratio can be used as a guide to the purity of the sample Some Instruments also contain factors for other common proteins such as BSA or IgG which allow users to choose the protein closest in type to their sample if the factor for the sample of protein is unknown 2 2 2 0 1 8 1 6 1 4 ie 1 0 0 8 0 6 0 4 0 2 0 0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 Wavelength nm Absorbance Fig 5 A typical wavelength scan for a protein sample 9 Protein concentration calculations The protein concentration c in mg ml is calculated by C Azgo E2801 mg mi X The absorbance coefficient E2801 mami corresponds to the Azgo of a 1 mg ml solution of the protein and varies between proteins E280 1 mg m CAN be determined 1 by measuring the absorbance of the protein in a solution of Known concentration or 2 by the theoretical calculation E280 1 mg ml 5500nrp T 1490Nyr T 125ns s M where Np Ntyr and ns s are the number of Trp and Tyr residues ns s is the number of disulfide bonds S S bonds in the protein sequence and M is the molecular weight of the protein Coenzymes and cofactors ma
32. using small volume cells or specialist small volume spectrophotometers Nucleic acid measurements with low volume instruments To get meaningful results as a rule of thumb the following two criteria must be met e AbS260 gt approx twice AbS239 A260 A230 Ratio 2 e AbS260 0 1 or more indicative of a high enough concentration ie solution not too dilute Typically the only measurements checked for DNA measurement are concentration and Az6o Azao ratios Unexpected results can often be explained by looking at the underlying Azz0 A260 A280 A320 Values As an example Sample 1 Sample 3 Concentration 10 7 ug ml Concentration 11 0 ug ml A260 A280 A320 A260 A280 A320 0 244 0 129 0 030 0 326 0 211 0 107 A260 A280 A260 A230 A260 A280 A260 A230 2 162 0 023 2 106 0 024 Sample 2 Sample 4 Concentration 11 1 ug ml Concentration 10 5 ug ml A260 A280 A320 A260 A280 A320 0 323 0 206 0 101 0 303 0 192 0 094 A260 Azso A260 Az30 Az6o Azao A260 A230 2 114 0 024 2 155 0 023 In this example the instrument is giving concentration values which are very close together lt 2 variation So the reproducibility for concentration values is within the expected values However looking at the underlying absorbance readings shows that the 230 nm reading is very high compared to other readings making the ratio Azgo Az30 very low A value lt 1 8 indicates potential contamination Note that in this example e The background at A329 nm
33. w Ready To Go SpinTrap Tricorn Whatman and Whatman GD X are trademarks of GE Healthcare companies TALON is a trademark of Clontech Laboratories Inc Ultrospec is a trademark of Biochrom Ltd Adobe is a registered trademark of Adobe Systems Incorporated in the United States Microsoft Windows and Vista are trademarks of Microsoft Corporation Bluetooth is a trademark of Bluetooth Special Interest Group SIG 2012 2013 General Electric Company All rights reserved First published October 2012 29 0331 82 AB 03 2013
34. y also contribute Examples of values for E280 1 mg ml include 0 67 for BSA 1 37 for IgG and 2 64 for lysozyme Light scattering correction of the Azgo value can be made by A280 Azag Measured 1 929 x A33 Measured Christian and Warburg 3 Nucleic acids have absorbance at 280 nm maximum at 260 nm If the presence of nucleic acids is suspected the protein concentration can be estimated with less accuracy according to Christian W and Warburg O 3 C mg ml 155 A280 0 76 x A650 The constants 1 55 and 0 76 refer to a specific protein used by Christian and Warburg For best accuracy the factors should be determined for the target protein at hand Refer to the NanoVue Plus User Manual 28 9574 75 from GE Healthcare Colorimetric methods A wide variety of colorimetic methods for protein concentration measurements are available they all work in a similar way The reagents contain a dye which binds specifically to the proteins in a solution The absorption of a known set of standards is measured and a standard curve produced Absorption measurements of unknown samples can then be compared to the curve to establish their concentration It is important to check the working ranges of the commercial product being used as these can vary 10 BCA 4 This method relies on the reaction of cupric ions Cu2 and peptide bonds This forms Cuprous ions Cu which are then detected with bicinchoninic acid BCA This give
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