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Section 10- Protein 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.

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Summary of Contents for ND-1000

Page 1: ...lmington DE 19810 USA 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 7 2007 ...

Page 2: ...rt F5 4 3 Show Report F7 4 3 Sample ID 4 3 Sample 4 3 Exit 4 3 Escape Key ESC 4 3 Show Context Help Ctrl H 4 3 User s Manual 4 3 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 Calcu...

Page 3: ...ord Kits Protocols and Sample Preparation 12 1 Unique Screen Features 12 2 Making Bradford Protein Measurements 12 2 Standard Curve Features 12 3 Delete Standard Points 12 3 Exiting the Bradford Module 12 4 13 Cell Cultures 13 1 Sample Size Requirements 13 1 Cell Suspension Concentrations 13 1 Sample Homogeneity 13 2 Decontamination of Measurement Pedestals 13 2 14 Archived Data and Data Viewer 14...

Page 4: ...Concentration Calculation Beer s Law 18 1 Solvent Compatibility 18 2 Decontamination of Measurement Optical Surfaces 18 2 Setting Up a Dymo 400 Label Writer Printer 18 2 ...

Page 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...

Page 6: ...view the contents of the CD Double click on the file 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 W...

Page 7: ...anoDrop ND 1000 Spectrophotometer while the instrument is not in use When the 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 fea...

Page 8: ...destals as shown above upon completion of each sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup Although generally not necessary 2 ul water aliquots can be used to clean the measurement 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 ...

Page 9: ... accuracy a 2 ul sample is recommended Sample Carryover Prevention of sample being retained on the ND 1000 Spectrophotometer s measurement pedestals is easily addressed Simple wiping of the upper and lower measurement pedestal with a dry laboratory wipe is highly effective in eliminating carryover for samples differing in concentration by as much as three orders of magnitude see data at www nanodr...

Page 10: ... Duplicate Data Folder Save the alternative path by 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 to select the Auto Reporting option for any...

Page 11: ...to both confirm that the instrument is performing within the pathlength calibration specifications and help troubleshoot operational problems with the instrument Note The inclusion of the calibration check utility is new to version 3 5 1 For more information on using this module refer to both section 15 Calibration Check and section 16 Troubleshooting of this manual Account Management The Account ...

Page 12: ...veral ways as noted below Failure to change password within the allotted time Incorrectly entering the password 99 consecutive times The administrator locks a specific account Only the administrator level 10 can unlock a locked account This is done by using the Modify User entry in the Account Management module Note All accounts even the administrator can be locked if the incorrect password entry ...

Page 13: ...ed dye methods are indicated by a diamond and can t be modified Note 2 Absorbance contribution at 260 nm and 280 nm from the respective dye can be corrected by entering the appropriate decimal correction field Refer to the dye manufacturer to find the 260 nm and 280 nm factor for dyes not pre defined in the Dye Chromophore List Note If upgrading from a version prior to 3 3 zero values 0 for 260 nm...

Page 14: ...ssing the F1 key or clicking the Measure button The entire measurement cycle takes approximately 10 seconds Blank F3 Before making a sample measurement 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 clea...

Page 15: ... 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 on any printer connected to the PC Print Window A Print dialogue can be initiated from the File pull down menu or by typing Ctrl P The user can specify any connected printer to print to from the Print dialogue Saving Current Screen as JPG Image The current screen can be ...

Page 16: ...e a flashing text cursor when the instrument is waiting to make a new measurement Sample The Sample indicator is activated when a sample report is being recorded It indicates the sample number of the last sample 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 T...

Page 17: ...Type used to select the color keyed type of nucleic acid being measured The user can select DNA 50 for dsDNA RNA 40 for RNA ssDNA 33 for single stranded DNA or Other for other nucleic acids The default is DNA 50 If Other is selected the user can select an analysis constant between15 150 When navigating amongst the three general sample types within the Nucleic Acids module the last constant value e...

Page 18: ...nce 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 very nearly zero absorbance All spectra are referenced off of this zero Spectrum Overlay Control The user can display more than one ...

Page 19: ...hat dye Note 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 you...

Page 20: ...esponding 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 calculations Dye 1 or 2 user selected dye Abs Norm normalized absorbance of selected Dye at the 1 mm pathlength pmol ul concentration based upon select...

Page 21: ... user selectable wavelength cursors and corresponding absorbencies for a 1 mm pathlength The wavelengths can be set 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 subt...

Page 22: ...ectable feature in this module If selected the software will automatically normalize the spectrum based on the lowest absorbance value in the range 400 750 nm This feature may be selected as the default option using the User Preferences module 7 2 ...

Page 23: ...ants 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 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 ra...

Page 24: ...ective protein reference Maximum value for e is 999 X 1000 and maximum value for M W is 9999 X 1000 User entered mass extinction coefficient L gm 1 cm 1 for a 10 mg ml 1 solution of the respective reference protein λ and Abs current value of the user selectable wavelength cursor and corresponding absorbance The wavelength can be set by dragging the cursor using the up down arrows or typing in the ...

Page 25: ...lear 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 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 Wh...

Page 26: ... 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 v...

Page 27: ...0 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 Concentration Calculation Beer s Law in the Appendices for more ...

Page 28: ...lumns break during measurement Additional information about the PR 1 kit may be found at www nanodrop com Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer the regular BCA assay can measure samples from 0 20 mg ml up to 8 0 mg ml A mini BCA assay covers an approximate range of 0 01 0 20 mg ml Assay Type Approx Lower Limit Approx Upper Limit Typical Reproducibility minimum 5...

Page 29: ...aded in the NanoDrop ND 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 ...

Page 30: ...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 Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Stan...

Page 31: ...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 4 ...

Page 32: ...pound 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 at www nanodrop com Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer the Modified Lowry assay can run from 0 20 mg ml up to 8 0 mg ml Assay Type Approx Lower Limit Ap...

Page 33: ...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 protein and a single replicate of one sta...

Page 34: ... 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 Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete Sta...

Page 35: ...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 4 ...

Page 36: ...id 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 at www nanodrop com Measurement Concentration Range On the NanoDrop ND 1000 Spectrophotometer using the regular Bradford assay unknown protein concentrations from 100ug ml up to several thousand micrograms ml u...

Page 37: ...t by the user Note The user selected wavelength and absorbance are not utilized in any calculations ug ml concentration of the sample unknown Making Bradford Protein Measurements A standard curve is required every time the Bradford assay is run Although curves can be saved and reloaded in the NanoDrop ND 1000 Spectrophotometer software it is recommended that the user follow manufacturers guideline...

Page 38: ...e 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 Curve functions Selecting the View Standard Curve button allows the user to review the standard curve at any time New to version 3 5 1 is the ability to reload a standard curve concentration series using the Load Standard Curve Set up Delete St...

Page 39: ... 8000 ug ml Note the linear range is 100 1000 ug ml A mini Bradford assay covers an approximate range of 15 100 ug ml Exiting the Bradford Module It is recommended that you process all of the unknowns to be assayed before exiting the Bradford software module 12 4 ...

Page 40: ... is displayed λ and Abs current value of the user selectable 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 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 th...

Page 41: ...s If decontamination is necessary a sanitizing solution such as a 0 5 solution of sodium hypochlorite 1 10 dilution of common commercial bleach solutions freshly prepared 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 Sol...

Page 42: ... path For MicroArray UV Vis BCA Protein Bradford Lowry and Cell Culture application modules the data are 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 va...

Page 43: ...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 important to exit out of the Data Viewer module and re enter if importin...

Page 44: ...Samples box Note The software defaults to a buffer size of 200 samples Search Function allows the user to locate specific data by searching through sample ID names Sample Information and Spectrum Populated with the information associated with the most recently highlighted sample Import and Return Uses selected sample data to populate Plots and Reports windows Note Holding down the shift or control...

Page 45: ...vable x and y axis Available for all data types If the cursor is out of view in either direction rescale the axis by typing over one of the outer limit numbers The cursor absorbance information displayed at the bottom of the page is determined by the position of the movable cursors The movable X determines the baseline from which the peak of the Y position is calculated Reset Baseline will reposit...

Page 46: ...ved in an ndv format The other two options are meant for reports that are expected to be opened in Excel type spreadsheets To open these reports go to the C NanoDrop Data Reports folder and right click on the file of interest Additional features of the Report page Test Type Automatically populated with data method type Date and time Auto fills in the date and time when a report is generated Report...

Page 47: ...iginal archive files are not altered during this process 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 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 ma...

Page 48: ...he CF 1 vial as directed in the image below Typically the target absorbance is 0 734 the actual value will depend on the lot of CF 1 3 Add 1ul of deionized water and select Blank 4 Before opening the ampoule of CF 1 Calibration Fluid shake vigorously to ensure solution is thoroughly mixed Ensure all solution is collected in the bottom portion of the ampoule 5 Carefully break the neck of the ampoul...

Page 49: ... 1 Calibration Fluid is supplied in a single use vial The CF 1 must be used within one hour of opening the vial Exposure to the environment or transferring of the fluid to another container may cause a significant change in concentration 15 2 ...

Page 50: ...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...

Page 51: ... are correct by opening the Power Options Properties page by choosing Start Æ Control Panel Æ Power Options 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 d...

Page 52: ...firm that the power supply is operating properly To do this connect the leads of a volt ohmmeter to the outlet of the supply The voltage should be 12 20 Vdc center positive If none of the troubleshooting steps above solve the problem refer to the Technical Service section for getting help from your distributor or NanoDrop Technologies Saturated Detector This error message can occur when the softwa...

Page 53: ... be purchased from VWR 2 Apply a very thin even layer of PR 1 to the surface of the upper and lower pedestals 3 Wait 30 seconds for the PR 1 to dry 4 Fold a clean dry laboratory wipe into quarters and remove the PR 1 by aggressively rubbing the surface of the upper and lower pedestals until all compound residue is removed Note The black appearance of the removed residue is normal The reconditionin...

Page 54: ...his folder Replace the files if they have been removed If the file cannot be located reinstall the software Other Software Error Messages Can t find file OOIDRV INI This error occurs when trying to install the software without administrator privileges Contact your system administrator to install the software Source Error This error indicates that there is insufficient light getting through to make...

Page 55: ...umn is formed If necessary try 1 5 2 ul samples to ensure the column is formed Also 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 sa...

Page 56: ... 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 when measuring the same nuclei...

Page 57: ... been created choose File Æ Save Window as 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 highlighte...

Page 58: ... removed Note Do not use a squirt bottle to apply de ionized water or HCl Calibration Wavelength The wavelength is auto calibrated based on known peaks in the xenon lamp spectra each time the software is started No calibration is required by the user Pathlength Accuracy Generally this is not required as field experience has shown that the path does not change appreciably even after several years o...

Page 59: ...red in memory as an array of light intensities by wavelength When 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 ...

Page 60: ...t Compatibility The NanoDrop ND 1000 Spectrophotometer is compatible with most solvents typically used in life 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...

Page 61: ...that the shipping label selection has been recognized by checking the set up from within the NanoDrop 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 the Dymo Printer under the Printer Name dropdown box 3 Next click on Properties and select Advanced on the next window 4 Select Paper size 30256 and clic...

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