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Section 10- Protein BCA

 

10. Protein  BCA 

 
The BCA (Bicinchoninic Acid) Protein Assay is an alternative method 
for determining protein concentration. It is often used for more dilute 
protein solutions and/or in the presence of components that also have 
significant UV (280 nm) absorbance. Unlike the Protein A280 method, 
the BCA Assay requires a standard curve to be generated each time it 
is run, before unknown proteins can be measured. The resulting Cu-
BCA chelate formed in the presence of protein is measured at its 
wavelength maximum of 562 nm and normalized at 750 nm. Pre-
formulated reagents of BCA and CuSO

4

, utilized in the assay, are 

available in kit form from numerous manufacturers. Follow their 
recommendations when mixing the respective reagents at the time 
the assay is to be performed.   
 
 

Sample Volume Requirements 

Some proteins are hydrophobic and others hydrophilic giving rise to 
variable surface tension properties in the sample to be measured.  
Additionally the presence of surfactants or detergents in reagents, 
such as the Bradford reagent, can significantly alter surface tension. 
This occurrence can be overcome without affecting the sample’s 
absorbance by using a larger sample volume. 

A 2 ul sample size is 

recommended for protein measurements

.  

 
 

Pedestal Reconditioning   

Solutions and reagents containing surfactants may “un-condition” the 
measurement pedestal surfaces so that the liquid column does not 
form.  If this occurs, “buff” the measurement pedestal surfaces by 
rubbing each measurement surface aggressively with a dry laboratory 
wipe 30-40 times.  This will “re-condition” the surface allowing the 
liquid sample column to form. Alternatively, use the NanoDrop 
Pedestal Reconditioning Compound (PR-1) as a rapid means of 
reconditioning the pedestals when the surface properties have been 
compromised and liquid columns break during measurement. 
Additional information about the PR-1 kit may be found on our 
website. 

 
 
Measurement Concentration Range 

When using a 20:1 reagent to sample volume dilution, the BCA assay 
concentration range of detection is ~0.20 mg/ml to 8.0 mg/ml on the 
NanoDrop 1000. Using a 1:1 reagent to sample volume dilution the 
concentration range of detection is 0.01 mg/ml – 0.20 mg/ml. 
 

10-1

 

Содержание NanoDrop 1000 V3.7

Страница 1: ...NanoDrop 1000 Spectrophotometer V3 7 User s Manual ...

Страница 2: ...rranties of merchantability and fitness for a particular purpose Customers are ultimately responsible for validation of their systems 2008 Thermo Fisher Scientific Inc All rights reserved No part of this publication may be stored in a retrieval system transmitted or reproduced in any way including but not limited to photocopy photograph magnetic or other record without our prior written permission...

Страница 3: ... blank F2 4 2 Print Screen F4 4 3 Start Report Recording 4 3 Print Report F5 4 4 Show Report F7 4 4 Sample ID 4 4 Sample 4 5 Exit 4 5 Escape Key ESC 4 5 Show Context Help Ctrl H 4 5 User s Manual 4 5 5 Nucleic Acids 5 1 Sample Volume Requirements 5 1 Measurement Concentration Range 5 1 Spectrum Normalization 5 3 Spectrum Overlay Control 5 3 6 MicroArray 6 1 Fluorescent Dye Selection 6 1 Sample Vol...

Страница 4: ...Features 11 5 Delete Standard Points 11 5 Exiting the Lowry Module 11 6 12 Protein Bradford 12 1 Sample Volume Requirement 12 1 Pedestal Reconditioning 12 1 Measurement Concentration Range 12 2 Bradford Kits Protocols and Sample Preparation 12 2 Unique Screen Features 12 3 Making Bradford Protein Measurements 12 3 Standard Curve Features 12 5 Delete Standard Points 12 6 Exiting the Bradford Module...

Страница 5: ...Ratio 17 11 Unusual Spectrum 17 13 Technical Support 17 13 18 Maintenance and Warranty 18 1 Cleaning 18 1 Calibration 18 1 Warranty 18 2 19 Appendices 19 1 Instrument Specifications 19 1 Blanking and Absorbance Calculations 19 1 Concentration Calculation Beer s Law 19 2 Solvent Compatibility 19 3 Decontamination of Measurement Optical Surfaces 19 3 Setting Up a Dymo 400 Label Writer Printer 19 3 ...

Страница 6: ...he gap between the fiber optic ends The gap is controlled to both 1mm and 0 2 mm paths A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample The instrument is controlled by PC based software and the data is logged in an archive file on the PC Applications UV VIS spectrophotometry is simple f...

Страница 7: ...1 Overview General UV Vis spectrophotometry Patents The sample retention technology used in the NanoDrop 1000 Spectrophotometer is covered under US patents 6 628 382 and 6 809 826 Other patents are pending 1 2 ...

Страница 8: ...allation WARNING The system software must be loaded onto the PC before the USB cable is connected Administrator access on the PC is required to install the software To properly install the operating software 1 Close all programs and make sure that the USB cable is unplugged 2 Insert the operating software CD in the CD drive of the PC The software installation menu should appear automatically If th...

Страница 9: ...on 3 Select the MS Sans Serif western font and select 8 point size 4 Click OK Choosing an alternative font may result in some text being truncated in the operating software window Software Upgrades Periodic upgrades are made to the operating software and are available for download See our website for the latest available software version Cable Connections To make measurements with the instrument c...

Страница 10: ...ng Your Instrument Please register your product We periodically update our software and add new features free of charge We would like to keep our user list updated so that we may alert you to these updates and all information supplied is completely confidential You can register your instrument on our website 2 3 ...

Страница 11: ...ystem are listed below 1 With the sampling arm open pipette the sample onto the lower measurement pedestal 2 Close the sampling arm and initiate a spectral measurement using the operating software on the PC The sample column is automatically drawn between the upper and lower measurement pedestals and the spectral measurement made 3 1 ...

Страница 12: ...edestals be cleaned thoroughly This will prevent the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low level measurements A final cleaning of all surfaces with de ionized water is also recommended after the user s last measurement Note Please do not use a squirt bottle to apply de ionized water Decontamination of Measurement Pedestals If ...

Страница 13: ...ell suspensions 1 2 ul It is best to use a precision pipettor 0 2 ul with precision tips to assure that sufficient sample 1 2 ul is used Lower precision pipettors 0 10 ul and larger are not as good at delivering 1 ul volumes to the measurement pedestal If you are unsure about your sample characteristics or pipettor accuracy a 2 ul sample is recommended Sample Carryover Prevention of sample being r...

Страница 14: ... result in a 1 2 increase in sample concentration This can be observed in the field by measuring the same sample successively over time Highly volatile solvents such as hexane will likely evaporate before the measurement can be completed Less volatile solvents such as DMSO can be used successfully Sample Recovery One of the advantages of the sample retention system is that samples can be recovered...

Страница 15: ...Acid concentration and purity of nucleic acid MicroArray dye incorporation concentration and purity of nucleic acid UV Vis general UV Vis measurements Cell Cultures absorbance light scattering measurement of suspended microbial cells Protein A280 concentration and purity of purified protein Proteins Labels concentration of dye labeled proteins conjugates and metalloproteins 3 5 ...

Страница 16: ... clicking on the Save and Exit button before exiting the User Preferences module The user may also elect to deselect an automatic prompt to close the Data Viewer whenever a module is closed The Data Viewer must be closed if a different module is opened before data can be reviewed Reports Users may choose the Auto Reporting feature which allows data to automatically populate the report for all samp...

Страница 17: ...are six sample type options available for purified protein analysis and concentration measurement The default setting is Other protein E1 See Section 8 for additional information about each sample type option Note Software versions 3 5 1 and higher include the option to select whether or not to have the data and spectrum normalized to the absorbance value at 340 nm Proteins and Labels There are si...

Страница 18: ...r whose initial password is nanodrop It is strongly recommended that the password be changed after initial account set up Any user can be set to a level 10 access although this is not recommended see Level 5 Note The administrator or the last level 10 user account may not be deleted Level 5 this is the security setting recommended for an ordinary user account An account with this access will be pa...

Страница 19: ...ANCEL the clock with reset and the user account and application module will remain active for another 4 hours If the time expires the open application module will close returning to the Main Menu and the Default user Account Lockout User specific accounts can become locked out in several ways as noted below Failure to change password within the allotted time Incorrectly entering the password 99 co...

Страница 20: ...ly copied to the c NanoDrop Data Log Files directory If for some reason these files are not copied automatically they must be manually copied from the C Program files NanoDrop version to the C Program files NanoDrop version directory Dye Chromophore Editor The Dye Chromophore Editor gives the user the ability to add their own dyes or chromophores in addition to the predefined fluorescent dyes avai...

Страница 21: ...Section 3 General Operation Note If upgrading from a version prior to 3 3 zero values 0 for 260 nm and 280 nm correction factors will be entered for all user defined dyes 3 11 ...

Страница 22: ...d the Measure button is inactive as noted by its grayed out appearance A blank must first be measured before the Measure button will become active The Measure button is used to initiate the measurement sequence for all samples non blanks It is actuated by depressing the F1 key or clicking the Measure button The entire measurement cycle takes approximately 10 seconds Blank F3 Before making a sample...

Страница 23: ...atory wipe 4 Analyze an aliquot of the blanking solution as though it were a sample This is done using the Measure button F1 The result should be a spectrum with a relatively flat baseline Wipe the blank from both measurement pedestal surfaces and repeat the process until the spectrum is flat See Blanking and Absorbance Calculations in the appendix for more information on blanking and absorbance c...

Страница 24: ...t dialogue Saving Current Screen as JPG Image The current screen can be saved as a jpg image file by selecting Save Window from the File pull down menu Start Report Recording The user can log measurement results in a report table and print them to the desired printer To initiate this feature select the Start Report button The default setting has the Recording feature activated Refer to Data Viewer...

Страница 25: ...Data and in a duplicate location if selected in User Preferences Note The system is configured to work with the Dymo Label Writer 400 printing on 30256 2 5 16 X 4 shipping labels but can print to any printer connected to the PC Show Report F7 The user can display the entries comprising the current Sample Report at any time by selecting the Show Report button This function will enable the Data View...

Страница 26: ...ement data is automatically saved to an archive file and requires no user action Escape Key ESC The escape key is set to exit out of all screens Hitting the escape key twice will log the user out of an application module Show Context Help Ctrl H Context Help is enabled in the Main Menu all function modules and the application modules The help feature is enabled by choosing Show Context Help from t...

Страница 27: ... your pipettor accuracy a 1 5 2ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample Measurement Concentration Range The NanoDrop 1000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng ul without dilution To do this the instrument automatically detects the high concentration and utilizes the 0 2mm pathlengt...

Страница 28: ...the up down arrows to the right of the wavelength box Note The user selected wavelength and absorbance are not utilized in any calculations A260 10 mm path absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length A280...

Страница 29: ... appreciably lower this may indicate the presence of co purified contaminants ng ul sample concentration in ng ul based on absorbance at 260 nm and the selected analysis constant See the Concentration Calculation Beer s Law in the appendix for more details on this calculation Spectrum Normalization The baseline is automatically set to the absorbance value of the sample at 340 nm which should be ve...

Страница 30: ...after each new report or accumulate plots until prompted to clear The Clear Now setting will clear all current and previous plots When the overlay function is active the software will auto scale the y axis based on the sample with the highest absorbance at 260 nm Note When the overlay function is active the Blank function does not clear the existing overlaid sample spectra 5 4 ...

Страница 31: ... Chromophore Editor button found in the main menu Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box The respective absorbance wavelength extinction coefficient and 260nm and 280nm corrections will be automatically utilized for measurement and concentration calculation The default settings remain Dye 1 set to Cy3 and Dye 2 set to Cy5 In addition to the fluoresce...

Страница 32: ...ibility minimum 5 replicates SD pmol ul CV Cy3 Cy3 5 Alexa Fluor 555 and Alexa Fluor 660 0 20 100 sample range 0 20 4 0 pmol ul 0 20 pmol ul sample range 4 0 pmol ul 2 Cy5 Cy5 5 and Alexa Fluor 647 0 12 60 sample range 0 12 2 4 pmol ul 0 12 pmol ul sample range 2 4 pmol ul 2 Alexa Fluor 488 and Alexa Fluor 594 0 40 215 sample range 0 40 8 0 pmol ul 0 40 pmol ul sample range 8 0 pmol ul 2 Alexa Flu...

Страница 33: ...on Calculation Beer s Law in the appendix for more details on this calculation λ and Abs Norm the user selected wavelength black cursor and corresponding absorbance at the 1mm pathlength The wavelength can be selected by dragging the black cursor or using the up down arrows in the wavelength box Note The user selected wavelength and absorbance at the 1 mm pathlength are not utilized in any calcula...

Страница 34: ...absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm See 260 280 Ratio in the Troubleshooting section for more detail...

Страница 35: ...r sample composition or your pipettor accuracy a 1 5 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path completely is covered by sample Measurement Concentration Range All NanoDrop 1000 spectrophotometers will measure absorbance up to the 10mm pathlength equivalent of 15 A NanoDrop 1000 Spectrophotometer instruments with serial numbers 500 or those that...

Страница 36: ... been field retrofitted This capability is selected by choosing the Hi Abs button on the header bar When this is selected the absorbance is measured using the short path 0 2mm and plotted as a red line normalized to a 0 1mm path for easier visual comparison Sample ID label will be stored with sample data in the file folder C NanoDrop Data User name HiAbs This data cannot be imported into the Data ...

Страница 37: ...ing adequate columns for measurement The column formation issue can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does not form If this occurs buff the me...

Страница 38: ...yed can be selected by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of the sample type box A description of each sample type is given below A general reference setting based on a 0 1 1 mg ml protein solution producing an Absorbance at 280 nm of 1 0 A where the pathlength is 10 mm or 1 cm Bovine Serum Albumin reference U...

Страница 39: ...ble wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cursor using the up down arrows or typing in the desired wavelength Note The user selected wavelength and absorbance are not utilized in any calculations A280 10 mm Path 10 mm equivalent absorbance at 280 nm for the protein sample measured A260 280 ratio of sample absorbance at 260 and 280 nm Spectrum Norm...

Страница 40: ...example above Spectrum Overlay Control The user can display more than one spectrum in the same display using this feature The current sample plot will be displayed in bold and previous plots will be distinguished by different colors as seen in the following example The default option is set to clear the display for the next reading The user may set the overlay control to clear after each sample pl...

Страница 41: ...ear Now setting will clear all current and previous plots When the overlay function is active the software module will auto scale the y axis based on the sample with the highest absorbance at 280nm Note When the overlay function is active the Blank function does not clear the existing overlaid sample spectra 8 5 ...

Страница 42: ... main menu Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box The respective absorbance wavelength extinction coefficient and 280nm corrections will be automatically utilized for measurement and concentration calculation The default settings remain Dye 1 set to Cy3 In addition to the fluorescent dyes available from the drop down menu an option entitled None is a...

Страница 43: ...allowing the liquid sample column to form Alternatively use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement Additional information about the PR 1 kit may be found on our website Measurement Concentration Range The NanoDrop 1000 Spectrophotometer will acc...

Страница 44: ... located to the left of the sample type box See Protein A280 Section 8 for a detailed description of each sample type λ3 user selected wavelength Norm Abs λ3 10mm equivalent normalized absorbance at the respective wavelength Dye 1 or 2 user selected dye Norm Abs λ1 normalized 10 mm equivalent absorbance of selected Dye uM concentration based upon selected Dye s extinction coefficient See the Conce...

Страница 45: ...table Baseline Type options The default setting is set to normalize the display spectrum at 750nm Alternatively the 400 750 Slope Baseline Type will normalize the display at 750 nm and accommodate any linear baseline offset across the 400 to 750 nm range The user may also elect whether or not to use a bichromatic normalization of the 280 nm absorbance value to the absorbance value at 340 nm 9 4 ...

Страница 46: ... in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Reconditioning Solutions and reagents containing surfactants may un condition the measurement pedestal surfaces so that the liquid column does n...

Страница 47: ...differences Note If you run the assay at 60 C doubling the volumes may afford greater insurance against skewed results from evaporation condensation within the sealed reaction tube Protein standards BSA for generating a standard curve may also be provided by the manufacturer for the BCA assay kit Follow the manufacturer s protocol for the assay including recommended incubation times and temperatur...

Страница 48: ...he NanoDrop 1000 Spectrophotometer software it is recommended that the user follow manufacturers guidelines and generate fresh standard curves for each assay Additionally a standard curve set up may be reloaded This feature will recall the respective standard series used in a previously saved standard curve Both single and multi point standard curve generation is incorporated into the software A s...

Страница 49: ...tions in the large text box on the right side of the screen Step 2 Measure Standards Up to 5 replicates of each of up to 7 standards can be measured The software will not allow measurement of samples until a minimum of 1 standard and references or 2 standards are measured Polynomial curve fitting requires more standard points 10 4 ...

Страница 50: ... Standard Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time Software versions 3 5 1 and higher include the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Standard Points The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the t...

Страница 51: ...BCA Regular BCA Standard Curve 0 2 8 0 mg ml mini BCA Standard Curve 0 01 0 20 mg ml Exiting the BCA Module It is recommended that you process all of the unknowns to be assayed before exiting the BCA software module 10 6 ...

Страница 52: ...ers hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larger sample volume A 2 ul sample size is recommended for protein measurements Pedestal Recon...

Страница 53: ... may also be provided by the manufacturer for the Modified Lowry assay kit Follow the manufacturer s protocol for the assay including recommended incubation times and temperature Additionally use the respective standard e g BSA and dilutions that cover the analytical range mg ml of interest Note Since the NanoDrop 1000 can measure higher protein concentrations than traditional cuvette based spectr...

Страница 54: ... manufacturers guidelines and generate fresh standard curves for each assay Additionally a standard curve set up may be reloaded This feature will recall the respective standard series used in a previously saved standard curve Both single and multi point standard curve generation is incorporated into the software A standard curve can be developed using a reference Modified Lowry reagent only no pr...

Страница 55: ...uctions in the large text box on the right side of the screen Step 2 Measure Standards Up to 5 replicates of each of up to 7 standards can be measured The software will not allow measurement of samples until a minimum of 1 standard and references or 2 standards are measured Polynomial curve fitting requires more standard points 11 4 ...

Страница 56: ...d Standard Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time Software versions 3 5 1 and higher include the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Standard Points The Delete Point button allows the user to delete points by first selecting the data point to be deleted in the ...

Страница 57: ...Section 11 Protein Lowry Modified Lowry Standard Curve 0 2 4 0 mg ml Exiting the Lowry Module It is recommended that you process all of the unknowns before exiting the Lowry software module 11 6 ...

Страница 58: ...iming throughout the assay Sample Volume Requirement Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured Additionally the presence of surfactants or detergents in reagents such as the Bradford reagent can significantly alter surface tension This occurrence can be overcome without affecting the sample s absorbance by using a larg...

Страница 59: ...mples unknowns is good practice particularly with the limited assay signal obtained with the Bradford Assay Assay Type Approx Lower Limit Approx Upper Limit Typical Reproducibility minimum 5 replicates SD ug ml CV Regular Bradford 100 ug ml 8000 ug ml sample range 100 500 ug ml 25 ug ml sample range 500 8000 ug ml 5 Mini Bradford 15 ug ml 100 ug ml sample range 15 50 ug ml 4 ug ml sample range 50 ...

Страница 60: ...t one standard before allowing measurement of samples Reference samples refer to the dye reagent without any protein added ie the 0 sample Replicate counter for tracking replicate number during reference and standard measurement Reset all Standards F11 clears all replicates of all standards Reset 1 Standard F12 clears all replicates of the selected standard Absorbance at 595 nm the protein dye com...

Страница 61: ... allows a maximum of five replicates for up to seven different standards There is no set order in which standards must be run A blank must be measured before the standard curve may be generated It is advisable to use water as the blank and use the dye reagent without any protein added as the 0 or reference sample There are three general procedural steps to unknown protein concentration measurement...

Страница 62: ...olation point to point between the two standards flanking the unknown sample or by using polynomial fitting Note In order to obtain a concentration value mg ml the sample unknown must fall within the limits of the standard curve Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard Curve pull down menu and choosing the Save as or Load Standard Cu...

Страница 63: ...and then choosing the Delete Point button If erroneous or non representative readings are encountered for a specific standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at once using the Reset all Standards button Regular Bradford curve covers 200 8000 ug ml Note the linear range is 100 1000 ug ml A mini Bradford assay cover...

Страница 64: ...Section 12 Protein Bradford Exiting the Bradford Module It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 12 7 ...

Страница 65: ... 5 over entire range 7 5 1 25 ug ml 1000 ug ml 5 over entire range A regular assay using a 15 1 reagent sample volume ratio To accurately prepare standards we suggest using a minimum sample volume of 4 ul in 60 ul of the Pierce 660 nm reagent larger sample volume is preferable A mini assay using a 7 5 1 reagent sample volume ratio To prepare sufficient volume of mixtures we suggest using a minimum...

Страница 66: ...cursor wavelength can be set by the user Note The user selected wavelength and absorbance are not utilized in any calculations ug ml concentration of the sample unknown Making Pierce 660 nm Protein Measurements A standard curve is required by the software every time the Pirece Protein 660 nm assay is run Although curves can be saved and reloaded in the NanoDrop 1000 Spectrophotometer software it i...

Страница 67: ... other colorimetric assays on the NanoDrop 1000 where it is recommended that water be used as the blank There are three general procedural steps to unknown protein concentration measurements The requisite order including generating the standard curve is as follows Step 1 Measure the Reference 660 nm reagent a zero Standard Note The software will guide the user with instructions in the large text b...

Страница 68: ...be calculated by using linear interpolation point to point between the two standards flanking the unknown sample or by using polynomial fitting Note In order to obtain a concentration value mg ml the sample unknown must fall within the limits of the standard curve Standard Curve Features Standard curves can be saved and reloaded for reference use by using the Standard Curve pull down menu and choo...

Страница 69: ... the table and then choosing the Delete Point button If erroneous or non representative readings are encountered for a specific standard all replicates of that standard are cleared by selecting Delete 1 Standard Additionally all standards can be deleted at once using the Reset all Standards button The 15 1 reagent to sample ratio assay covers 500 2000 ug ml 13 5 ...

Страница 70: ...ed using classical cuvette based systems will be attributable to the shorter pathlength 1 mm vs 1 cm Values may not be exactly 10 fold different as readings are dependent on both the optics of a specific spectrophotometer as well as the cell type in suspension The Cell Cultures module displays the sample spectrum from 250 nm to 700 nm One cursor is fixed at the frequently used wavelength for monit...

Страница 71: ...spension Concentrations Due to its shorter path length the NanoDrop 1000 Spectrophotometer can measure absorbencies that are 10 fold higher than those measurable on a standard cuvette spectrophotometer This makes it possible to directly monitor concentrated cell suspensions Since the entire spectrum is displayed diluted samples exhibiting very low Absorbance at 600 nm can be monitored at lower wav...

Страница 72: ...Section 14 Cell Cultures Compatibility appendix Note Please do not use a squirt bottle to apply either a cleaning solution or water to the pedestal 14 3 ...

Страница 73: ...description of Data Viewer below The data may be edited and or reformatted and stored under names of the user s choice The spectrum can be re plotted from the wavelength data if needed for further analysis Note1 Absorbance data shown in archive files are represented as they are displayed on the screen For Nucleic Acids Protein A280 and Protein and Labels application modules data are stored based o...

Страница 74: ...e their data to an additional location This option can be chosen under the Archiving Tab in User Preferences on the Main Menu by setting the Duplicate data storage box to On and then choosing the file path by clicking on the file folder icon under Duplicate Data Folder Save the alternative path by clicking on the Save Preferences button before exiting the User Preferences window All data are writt...

Страница 75: ...ures common to all three pages include File Options controlled by this tool bar function include File Page Set up Print Window and Save Window Configuration Options controlled by this tool bar function include Auto Scale Include graph in printout and Include standards in printout Data Includes options to import data rename samples and delete sample data Note After deleting all samples it is import...

Страница 76: ...f each file name will provide further detail to each level Users may choose to select either individual samples within a file or the entire file All import selections must be of the same application or method type Note Hi Abs data cannot be imported into the Data Viewer but can be opened with Excel type spreadsheets Archive File Converter Data generated with earlier versions of the operating softw...

Страница 77: ...he most recently highlighted sample Import and Return Uses selected sample data to populate Plots and Reports windows Note Holding down the shift or control PC function keys will allow the user to select multiple samples and or files for importing The keys can also be used to deselect multiple samples as seen in the following example Plots The Plots page displays selected sample spectra Plot Featu...

Страница 78: ... to a plot color The user is not able to select or highlight a sample from the legend Sample information Automatically populates with data associated with selected sample Data displayed is appropriate for data type chosen Note Information is based on data collected at the time the sample was measured and is not modified by a change in cursor position on the Data Viewer real time display Movable x ...

Страница 79: ... the reports page displaying only the columns of interest Report Features include Sort Allows users to sort data by column example by date or sample name and by either ascending or descending order Save Report Format Saves the current report format as an ndf file for retrieval and future use To designate a saved report format as the default format exit to the Main Menu choose Users Preferences and...

Страница 80: ...the Data Viewer to reload the report at a later date The saved report may be recalled using the pull down Load Report If using the Load Report feature the report will be displayed with the default column configuration Note Access the User Preferences module on the main menu to modify and save preferred default configurations Reports are saved in an ndv format The other two options are meant for re...

Страница 81: ...t Note This page is available for software modules utilizing a Standard Curve Archive File Converter All archive files located in the c Nanodrop Data folder generated with earlier versions of the operating software will automatically be copied and converted to version compatible ndj files upon first use of the software Archive files generated with earlier versions of software and not stored in the...

Страница 82: ...b delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program When opening with Excel be sure to save the file as a new name before making any changes to the data If changes are made to the data in the ndj file the Data Viewer module may not be able to recognize and import the data 15 10 ...

Страница 83: ...ions used to confirm calibration of standard spectrophotometers and is available through Thermo Fisher Scientific or your local distributor 2 2 7 2 2 7 Procedure 1 Ensure the measurement pedestals are clean and that a 1ul water sample beads up on the lower pedestal 2 Remove any lint build up from around the instrument solenoid See the instructions at the end of this section for removal of lint bui...

Страница 84: ...measure 10 replicates After the 10th measurement the calibration check results will be displayed on screen in the Customer Guidance text box 9 If the instrument does not pass the calibration check using 1ul samples immediately repeat the procedure again step 6 using 2ul samples A table of the Calibration Check tolerances and specifications is available through the Help menu drop down To print a co...

Страница 85: ... Some brands of lab wipes shred during the process and may result in a build up of lint under the instrument solenoid A significant build up of lint may alter the absorbance pathlength resulting in erroneous measurements Refer to the image and follow the instructions below to remove this lint build up 1 Lay the instrument on its side with the source fiber black fiber optic cable facing up 2 Open t...

Страница 86: ... Follow the onscreen instructions from USB Reset After unplugging and then reconnecting the USB cable the Found New Hardware Wizard should start as shown below Windows XP SP2 operating system will ask to allow it to search the internet for the proper software as shown Select No not this time Follow the prompts for automatic installation of the software Intro Page Windows XP SP2 Other Windows Opera...

Страница 87: ... the idVendor and idProduct are different than below or if no USB device is present in the list go to step 7 7 Install the instrument on another PC to rule out a faulty USB hub port on the original PC Run USBVIEW on the 2nd PC If the error continues contact your local distributor or Technical Support Connection Error This error occurs whenever the USB connection is disrupted while operating a soft...

Страница 88: ...ptions The System Standby and System Hibernate should be set to Never for the Plugged In column as shown below Static Electricity Discharge Discharge from the user to the instrument can be a problem in very dry environments It may be necessary for the user to wear a grounding strap to prevent the discharges from occurring Defective USB Port on PC If your instrument operates properly most of the ti...

Страница 89: ...select Intensity Check You should see a red and black spectrum and a bias value greater than 65 as shown below This indicates that the USB communication is normal the power supply is operational and the flash lamp is functioning If no spectra appear in the image confirm that the power supply is firmly connected to the instrument and the plug is connected to a working outlet Next confirm that the p...

Страница 90: ...low 1 Apply 5 ul of dH20 solution onto each bottom pedestal 2 Lower the upper pedestal arm to form a liquid column let it sit for approximately 2 3 minutes 3 Wipe away the water from both the upper and lower pedestal with a clean lab wipe Note Typically dH20 is sufficient for removal of samples that have dried on the optical pedestals There are a few cases i e dried proteins that may require a mor...

Страница 91: ...id Column Breakage This warning occurs when a possible problem with the column is detected The software compares the long path and short path absorbances and issues a warning to the user if the short path is not 20 of the long path absorbance within a tolerance The most common explanation is that the column is not forming properly due to the pedestal being unconditioned When a pedestal becomes unc...

Страница 92: ... is complete once the laboratory wipe shows is clean of black residue To check the effectiveness of the reconditioning load a 1 ul aliquot of dH20 onto the lower measurement pedestals and visually verify that the water beads up Additional information about the PR 1 kit may be found on our website As an alternative to using the PR 1 Kit the pedestals may be reconditioned as follows 1 Fold a clean d...

Страница 93: ...e software components have been removed or corrupted If this occurs reinstall the Labview Runtime engine by selecting Start Æ ProgramsÆ NanoDropÆUtilitiesÆ Runtime Installer Report Format Loading Error This error occurs when the user does not have Write access to one of the report format files located at C Nanodrop data custom report formats or the file has been moved from this folder Contact your...

Страница 94: ... to the bottom and not along the side Low Detector Bias This occurs when the software has detected a problem with the detector Contact your local distributor or Technical Support if you encounter this error OOIDRV Timeout This error occurs when a necessary file is deleted as a previous version of the software is uninstalled Reinstall the newest software to correct the problem EZUSB SYS Cannot Be F...

Страница 95: ...actice to clean the sample surfaces with de ionized water before opening an acquisition module to remove any dried down sample that might be present Note Do not use a squirt bottle to apply de ionized water Use a 1 5 2 ul sample size Very strange results can occur when the liquid sample column is not completely formed during a measurement While making a measurement visually confirm that the liquid...

Страница 96: ...pe the blank from both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum is within 0 005 A 1mm path Confirm that reference blank solution and solvent are the same material Some buffer components absorb in the UV range and therefore it is critical to blank the instrument with exactly the same material that the sample is suspended in Confirm that your sam...

Страница 97: ...60nm is generally on a plateau the absorbance curve at 280nm is quite steeply sloped A slight shift in wavelength accuracy will have a large effect on 260 280 ratios For example a 1 nm shift in wavelength accuracy will result in a 0 2 change in the 260 280 ratio Since many spectrophotometers claim a 1 nm accuracy specification it is possible to see as much as a 0 4 difference in the 260 280 ratio ...

Страница 98: ...saturated detector are shown below Detector saturation nucleic acid measurement Detector saturation Bradford measurement A spectrum that is very un smooth or ragged can be caused by insufficient light intensity reaching the spectrometer If you suspect that this is occurring contact your local distributor or Technical Support for assistance Technical Support If after referring to the above troubles...

Страница 99: ...Word MS Paint this program usually comes standard with the PC and can usually be found in the Start Æ Accessories menu or other graphics programs Save this as a jpg or doc file and send as email attachment to your local distributor or Technical Support Data Archive Files If you have questions about your data please send the archive file containing the suspect data as an email attachment to your lo...

Страница 100: ...are a few cases i e dried proteins that may require a more rigorous cleaning protocol For these cases we recommend that 0 5M HCl be substituted for the 5 ul of dH20 Please follow an HCl application with 5 ul of dH20 to ensure any residual HCl is removed Note Do not use a squirt bottle to apply de ionized water or HCl Calibration Pathlength Accuracy Calibration Check It is good practice to verify t...

Страница 101: ...ty All NanoDrop spectrophotometers and accessories manufactured by Thermo Fisher Scientific are warranted against manufacturing defects in parts and labor for a period of one year Preventive Maintenance as well as additional one two and three year warranty extensions are available More information about the various plans may be found on our website 18 2 ...

Страница 102: ... Voltage 12 Vdc Operating Power Consumption 6 W Standby Power Consumption 1 5 W CE Approval Units sold in Europe Australia and New Zealand UL CSA Approval Units sold in N America S America Asia and Africa Included in system software compatible with Windows 2000 or XP Blanking and Absorbance Calculations When the NanoDrop 1000 Spectrophotometer is blanked a spectrum is taken of a reference material...

Страница 103: ...ach dye is below Dye Type Extinction Coefficient liter mol cm Measurement Wavelength nm Cy3 150000 550 Cy5 250000 650 Alexa Fluor 488 71000 495 Alexa Fluor 546 104000 556 Alexa Fluor 555 150000 555 Alexa Fluor 594 73000 590 Alexa Fluor 647 239000 650 Alexa Fluor 660 132000 663 Cy3 5 150000 581 Cy5 5 250000 675 Nucleic Acids For nucleic acid quantification the Beer Lambert equation is modified to u...

Страница 104: ...nol acetone ether chloroform carbon tetrachloride DMSO DMF Acetonitrile THF toluene hexane benzene sodium hydroxide sodium hypochlorite bleach dilute HCl dilute HNO3 dilute acetic acid All forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable Decontamination of Measurement Optical Surfaces If decontamination is necessary a sanitizing solution...

Страница 105: ... this window then click Apply 8 Click on the Device Settings tab and ensure 30256 Shipping Label is selected as the Default label It is best to confirm that the shipping label selection has been recognized by checking the set up from within the operating software 1 Open the Data Viewer module and select Page Set up under the File drop down menu 2 Click on the Printer Set up button and then select ...

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