Thermo Scientific NanoDrop 1000 V3.3 Скачать руководство пользователя | Manualshive

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Section 9- Proteins & Labels

9. Proteins

&

Labels

This software module can be used to determine protein concentration (A280nm) as well as fluorescent dye concentration (protein array
conjugates), or to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios.

Fluorescent Dye Selection

There are currently nine fluorescent dyes that are hard-coded for use with the Proteins and Labels module (see table below). Users can
also enter & save fluorescent dyes not coded within the ND-1000 software using the ‘Dye/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 280nm % corrections will be automatically utilized for measurement and concentration calculation. The
default settings from NanoDrop remain Dye 1 set to Cy3 and Dye 2 set to Cy5. In addition to the fluorescent dyes available from the
drop-down menu, an option entitled ‘None’ is also available. Selecting ‘None’ disables the respective calculations & numeric displays
corresponding to that dye.

Note: Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes.

Sample Volume Requirements

Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the samples 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.

Special Cleaning Requirements for Proteins

Proteins and solutions containing surfactants are known to “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 with a dry laboratory wipe 15-20 times. This
will “re-condition” the surface allowing for the liquid sample column to form.

Measurement Concentration Range

The NanoDrop

®

ND-1000 Spectrophotometer will accurately measure protein samples up to 20 mg/ml (BSA) without dilution. A table of

concentration range and typical reproducibility is listed below.

Sample

Type

Detection

Limit

Approx.

Upper

Limit

Typical Reproducibility

(minimum 5 replicates)

(SD= mg/ml; CV= %)

Purified BSA

0.10 mg/ml

20 mg/ml

sample range 0.05-10 mg/ml:

±

0.10 mg/ml

sample range >10mg/ml:

±

2%

Cy3

0.2uM

100uM

sample range 0.20-4.0 pmol/ul:

±

0.20 pmol/ul

sample range >4.0 pmol/ul:

±

2%

Unique Screen Features

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Содержание NanoDrop 1000 V3.3

Страница 1: ...ND 1000 Spectrophotometer V3 3 User s Manual ...

Страница 2: ...Voice 302 479 7707 Fax 302 792 7155 Email info nanodrop com www nanodrop com NanoDrop is a registered trademark of NanoDrop Technologies Inc Other parties trademarks are the property of their respective owners and should be treated as such Copyright 2007 NanoDrop Technologies Inc rev 3 2007 ...

Страница 3: ...ording F6 4 7 Print Report F5 4 7 Show Report F7 4 8 Sample ID 4 8 Sample 4 8 Exit 4 8 Show Context Help 4 8 User s Manual 4 8 5 Nucleic Acids 5 1 Sample Volume Requirements 5 1 Measurement Concentration Range 5 1 Spectrum Normalization 5 2 Spectrum Overlay Control 5 2 6 MicroArray 6 1 Fluorescent Dye Selection 6 1 Sample Volume Requirements 6 1 Measurement Concentration Range 6 1 Baseline Calcula...

Страница 4: ...que Screen Features 12 1 Making Bradford Protein Measurements 12 2 Standard Curve Features 12 3 Exiting the Bradford Module 12 3 13 Cell Cultures 13 1 Sample Size Requirements 13 1 Cell Suspension Concentrations 13 1 Sample Homogeneity 13 1 Decontamination of Measurement Pedestals 13 2 14 Archived Data and Data Viewer 14 1 Archive File Creation 14 1 Data Storage Hierarchy 14 1 Data Viewer 14 2 Arc...

Страница 5: ...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 special software run from a PC and the data is logged in an archive file on the PC Applications UV VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop ND 1000 Spectrophotometer The small sample...

Страница 6: ...le named nd 1000 install exe 3 After software installation connect the USB cable and 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 All Windows Operating Systems Your N...

Страница 7: ...he unit is in this standby mode power consumption is 1 5 W and the flashlamp is not energized Also the instrument does not utilize a power switch or give a visual indication of the operability of the 12V power supply Registering 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...

Страница 8: ...surfaces after particularly high concentration samples to ensure no residual sample is retained on either pedestal After measuring a large number of samples however it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly This will prevent the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low level measu...

Страница 9: ... optic cable There are no cracks or crevices for residual sample to get trapped within Sample Homogeneity Sampling from non homogeneous solutions particularly when using small volumes can cause significant deviations in the data generated using all measurement technologies including spectrophotometry Genomic DNA lambda DNA and viscous solutions of other highly concentrated nucleic acids such as re...

Страница 10: ...fore exiting the User Preferences module Auto Reporting Users may choose to select the Auto Reporting option for any of the application modules The auto reporting option allows data to automatically be saved to the report for all samples Users may choose this option under the Report tab by selecting the corresponding box next to the modules listed under Auto Reporting Save the auto reporting funct...

Страница 11: ...count by using the pull down menu to select either Default or another user name or 2 the user closes the software A user account may also be logged out automatically if the software System Idle Timeout is exceeded After 4 hours of inactivity the software account will automatically revert back to the Default user A screen will appear indicating that the time is about to expire with a 30 second coun...

Страница 12: ... program files NanoDrop version to the c program files NanoDrop V 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 available for use with the MicroArray and Proteins and Labels modules Note 1 Predefined dye methods are indicated by a diamond and can t be modified Note ...

Страница 13: ...easurement a blank must be measured and stored see Blanking and Absorbance Calculations in the appendix for more details on absorbance calculations After making an initial blank measurement a straight line will appear on the screen subsequent blanks will clear any sample spectrum and display a straight line as shown in the image below Blanking Cycle For the most consistent results it is best to be...

Страница 14: ...e pull down menu Start Report Recording F6 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 section 14 Data Viewer for additional details Note To override this feature click on the Recording button Once de selected the button will read...

Страница 15: ...processed in the current report and increments with each successive measurement until the sample report is fully populated The sample buffer limit can be modified on the report page Exit This command closes all application modules and supporting options After clicking the Exit button the user has 10 seconds to cancel the exit command If no action is taken within 10 seconds the exit command is carr...

Страница 16: ...or more details on this calculation λ and Abs the user selected wavelength and corresponding absorbance The wavelength can be selected by moving the cursor or using the up down arrows to the left of the wavelength box Note The user selected wavelength and absorbance are not utilized in any calculations A260 absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm pat...

Страница 17: ...nt sample plot will be displayed in bold and previous plots will be distinguished by different colors See example below 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 plot default setting after each new report or accumulate plots until prompted to clear The Clear Now setting will clear all current and previous plo...

Страница 18: ... Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes Sample Volume Requirements Field experience has indicated that 1 ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous nucleic acid samples containing incorporated fluorescent dyes However if you are unsure about the surface tension properties of your sample or ...

Страница 19: ...tion Calculation Beer s Law in the appendix for more details on this calculation ng ul concentration of nucleic acids in the sample calculated using the absorbance at 260 nm minus the absorbance at 340 nm i e normalized at 340 nm and the nucleic acid analysis constant See Concentration Calculation Beer s Law in the appendix for more details on this calculation Show Report formatted for 200 samples...

Страница 20: ...by dragging the cursor using the up down arrows or typing in the desired wavelength Baseline the absorbance of the user selectable baseline horizontal cursor The user may drag this cursor to a new vertical position to create a new baseline The absorbance value of the baseline is subtracted from the absorbance of the spectrum Max Absorbance used to rescale the upper limit of the vertical axis Hi Ab...

Страница 21: ...occurs buff the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15 20 times This will re condition the surface allowing for the liquid sample column to form Measurement Concentration Range The NanoDrop ND 1000 Spectrophotometer will accurately measure protein samples up to 100 mg ml BSA without dilution To do this the instrument automatically detects the high concentration...

Страница 22: ...protein sample measured A260 280 ratio of sample absorbance at 260 and 280 nm Show Report formatted for 200 samples although the buffer size can be increased to accommodate thousands of samples Spectrum Normalization The baseline is automatically set to the absorbance value of the sample at 340 nm which should be very nearly zero absorbance All spectra are referenced off of this zero All data are ...

Страница 23: ...r user entered dyes Sample Volume Requirements Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the samples 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 sa...

Страница 24: ...λ1 normalized 10 mm equivalent absorbance of selected Dye uM concentration based upon selected Dye s extinction coefficient See the Concentration Calculation Beer s Law Appendix for more details on this calculation Abs ratio λ1 λ3 ratio of the absorbance of Dye 1 to the absorbance at the user selected wavelength λ3 mg ml concentration of proteins in the sample calculated using the absorbance at 28...

Страница 25: ... ml over entire range BCA Kits Protocols and Sample Preparation Commercial BCA Protein kit manufacturers typically outline procedures for two different protein concentration ranges A regular assay using a 20 1 reagent sample volume ratio To accurately prepare standards we suggest using a minimum sample volume of 4 ul in 80 ul of BCA reagent larger sample volume is preferable A mini assay using a 1...

Страница 26: ...light indicates the standard curve is incomplete and not yet ready for sample measurements Step 2 Measure Standards Up to 5 replicates of each standard 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 depending on the polynomial degree selected Step 3 Me...

Страница 27: ...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 exciting the BCA software module 10 3 ...

Страница 28: ...Upper Limit Typical Reproducibility minimum 5 replicates CV Modified Lowry 0 2 mg ml 4 0 mg ml 2 over entire range Modified Lowry Kits Protocols and Sample Preparation Commercial Modified Lowry Protein kit manufacturers typically outline procedures for their assays Modified Lowry assay using a 5 1 reagent sample volume ratio To accurately prepare Standards we suggest using a minimum sample volume ...

Страница 29: ...dicates the standard curve is incomplete and not yet ready for sample measurements Step 2 Measure Standards Up to 5 replicates of each standard 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 depending on the polynomial degree selected Step 3 Measure Sa...

Страница 30: ...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 exciting the Lowry software module 11 3 ...

Страница 31: ...dye and Coomassie dye protein aggregates are frequently encountered in Coomassie dye based protein assays With time particulate can be observed which can cause significant fluctuations in Absorbance readings It is also important to note the total analyte protein dye signal at 595nm is limited to 0 150 A as a result of the 1 0mm pathlength of the instrument the Bradford Coomassie dye reagent concen...

Страница 32: ...ximum of five replicates for each of seven different standards There is no set order in which standards must be run There are only three general procedural steps to unknown protein concentration measurement The requisite order including generating the standard curve is as follows Step 1 Measure the Reference Bradford reagent a zero Standard The software will not allow measurement of samples until ...

Страница 33: ...in 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 Reset this std Additionally all standards can be deleted at once using the Reset This Window F11 button Regular Bradford curve covers 200 8000 ug ml Note the linear range is 100 1000 ug ml A mini Bradford...

Страница 34: ...entrations Due to its shorter pathlength the ND 1000 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 wavelengths for example 280 nm Unique Scr...

Страница 35: ...chlorite bleach freshly prepared or other decontaminating solutions can be used to ensure that no biologically active material is present on the measurement pedestals The metal fiber optic fittings are made from 303 stainless steel and are very resistant to most common laboratory solvents see Solvent Compatibility appendix 13 2 ...

Страница 36: ...ation modules the data is normalized to a 1 0 mm 0 1 cm path For high absorbance UV Vis samples data are stored based on a 0 1 mm path Note 2 For data from all modules a column entitled Measurement Type is included For each measurement this column will contain Measure Blank or Reblank If the value is Measure then the values in that row are from a normal measurement that has utilized the stored bla...

Страница 37: ... the user to define the page set up for printing out the spectra the report and the standard curve Print Window The current Plot Report or Standards screen may be printed by selecting Print Window or CTL P Save Window Saves files as JPGs Help Context Help This feature is enabled in the Main Menu all function modules and the application modules The help feature is enabled by choosing Show Context H...

Страница 38: ... the legend The selected sample will show up as a bold plot line Plots Sets Users may select the maximum number of individual plots up to 20 graphed per page Since a report can hold data for many samples and a graph page is limited to 20 plots additional sample spectra are displayed on new pages Each page is then referred to as a set Legend Positioning the cursor over the legend box will bring up ...

Страница 39: ...ther 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 click Reports Use the Select Default Report Format to see the list of saved formats available for the specific method type Load Report Format Allows saved report ...

Страница 40: ...eport Buffer mode Drop down box defining options for managing reports Max Buffer size Default number is set at 200 When the selected buffer size is reached the Report will either be Saved Cleared or Printed and Cleared Standards Page The Standards page will display the actual reference standards applied to each particular sample at the time of measurement Note This page is available for software m...

Страница 41: ...n 14 Archived Data and Data Viewer Opening Archived Data with Spreadsheet Programs The archived files are in tab delimited format and can be opened in Microsoft Excel or an equivalent spreadsheet program 14 6 ...

Страница 42: ...nstallation of the software Intro Page Windows XP SP2 Other Windows Operating Systems 4 Try the NanoDrop software if it works properly you are finished If it does not operate properly go to step 5 5 Shutdown the NanoDrop software and open the USBView utility to confirm proper USB communication Start Æ Programs Æ NanoDrop Æ Utilities Æ USBView If USBView is not installed on your PC you can download...

Страница 43: ...ndby 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 time but the Connection...

Страница 44: ... distributor or NanoDrop Technologies Saturated Detector This error message can occur when the software calculates too high integration times during initialization This is most likely due to a dried sample left on the measurement surface after the last use of the instrument Cleaning both the top and bottom pedestals with de ionized water and exiting out of the software module to the main menu shou...

Страница 45: ...contact NanoDrop Technologies or your local distributor Error Code 8 This error code is most likely to occur if the Windows account that is currently logged into Windows does not have read and write access to the folder c nanodrop data or one of its subfolders See your PC administrator to make sure that all users of the NanoDrop software operate with a Windows account that has the appropriate acce...

Страница 46: ...e it could be the result of sample or aliquot non homogeneity or liquid column breakage It may be helpful to try the following to ensure representative results Make sure the sample surfaces are clean before starting the software module A dirty sample pedestal on startup can cause erroneous absorbance readings even negative values and signal saturation It is always a good practice to clean the samp...

Страница 47: ...illiam W Wilfinger Karol Mackey and Piotr Chomczynski Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity BioTechniques 22 474 481 March 1997 Wavelength accuracy of the spectrophotometers Although the absorbance of a nucleic acid at 260nm is generally on a plateau the absorbance curve at 280nm is quite steeply sloped A slight shift in wavelength accuracy wil...

Страница 48: ...s shown below Save to your hard drive and email as an attachment to your distributor or to info nanodrop com Application Module Screen Captures Screen captures of the actual spectrum as seen on your PC are of great use in diagnosing problems Making a screen capture is quite easy When in an application module press Alt Print Screen This copies the highlighted screen window to the PC s clipboard Nex...

Страница 49: ... not change appreciably even after several years of heavy use However it is good practice to check the calibration every six months using CF 1 calibration fluid A calibration check procedure is available from the Downloads section on the Support tab at www nanodrop com Parts That Require Replacement In general the only part that should periodically require replacement is the flash lamp The flash l...

Страница 50: ...hen a measurement of a sample is taken the intensity of light that has transmitted through the sample is recorded The sample intensities along with the blank intensities are used to calculate the sample absorbance according to the following equation Absorbance log Intensitysample Intensityblank Thus the measured light intensity of both the sample and of the blank are required to calculate the abso...

Страница 51: ...e science laboratories These include methanol ethanol n propanol isopropanol butanol 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 M...

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