Qubit® Protein Assay Kits
Contents
1. Qubit 2 0 Fluorometer above for calibration guidelines On the Standards Screen select to run a new calibration or to use the last calibration Press Yes to run a new calibration then Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid then press Read The reading takes approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid then press Read Remove Standard 2 Insert the tube containing Standard 3 in the Qubit 2 0 Fluorometer close the lid then press Read Remove Standard 3 OR Press No to use the last calibration The Sample Screen is displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid then press Read After the measurement is completed the result is displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the concentration of your sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator on page 5 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample then press Read Next Sample Repeat sample readings until all samples have been read The Qub2. hours Common contaminants such as reducing reagents DTT B mercaptoethanol salts free nucleotides amino acids solvents or DNA but not detergents are well tolerated in the assay some very slight modifications for the procedure are required for other contaminants Appendix Table 2 In addition to the Qubit Protein Assay Kits described here we also offer other kits for assaying dsDNA and RNA Appendix Table 3 MANO0002349 Revised 4 August 2011 MP 33211 Before You Begin Materials required but not provided Storing the Qubit Protein Assay Kits Critical assay parameters e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit protein reagent and buffer are designed for room temperature storage The Qubit protein reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the protein standards at 4 C Assay temperature The Qubit protein assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit protein reagent and the Qubit protein buffer at room temperature a
3. 00 ug mL OKt DNA 10 tt 10 tt 10 tt OK DNA 50 tt 50 tt 50 tt NR BSA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approximate in 20 uL or 10 uL sample volumes are also listed Results are given as OK usually lt 10 perturbation or as NR not recommended tAn acceptable result but with some distortion of the standard curve For best results add the same amount of contaminant to the standard samples A precipitate was observed A mixture of 19 amino acids A mixture of dATP dCTP dGTP and dTTP ttFor each data point the DNA mass was a fixed percentage of the protein mass For accurate results add the same amount of PBS to standard samples Qubit Protein Assay Kits 7 Qubit Assay Kits compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 can help you choose a kit based on the target molecule being measured and the number of assays you require Product Catalog no numberof Target Notes assays Qubit dsDNA BR Assay Kit Q32850 100 Core range high confidence 0 01 g mL to 5 ug mLt Extended range moderate confidence 5 ug mL to 10 ug mLt dsDNA Useful for quantitation of genomic and miniprep DNA samples Accurate
4. 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube then mix by vortexing 2 3 seconds Be careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit protein standard is added to 190 uL of Qubit working solution Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere from 1 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere from 180 199 UL Add each of your samples to the assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 then mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Qubit Protein Assay Kits 3 1 8 1 9 1 10 1 11 1 12 Calculating the concentration of your sample Allow all tubes to incubate at room temperature for 15 minutes On the Home Screen of the Qubit 2 0 Fluorometer press Protein The Standards Screen is displayed Note If you have already performed a calibration for the selected assay the Qubit 2 0 Fluorometer prompts you to choose between reading new standards and using the previous calibration See Calibrating the
5. g ovalbumin chicken egg lysozyme bovine milk B casein equine myoglobin bovine milk a casein porcine pepsin mouse immunoglobulin IgG and calf thymus histone Fluorescence was measured at 485 590 nm and plotted versus the mass of the protein sample At 3 yg the fluorescence variation was 12 4 or 8 7 excluding the basic histone protein Background fluorescence has not been subtracted Qubit Protein Assay Kits 5 Effect of temperature on the Qubit Protein Assay 160 14099 amp 140 go 2 Q 120 F PZ 100 QE 8 80 De Q 60 38 oN 40 aac E 20 e amp 0 r r r r 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence versus temperature for the Qubit protein assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay How the Qubit 2 0 Fluorometer calculates concentration 120 Standards 100 4 Raw Data Curve Fitting Algorithm L Relative fluorescence D O oO oO to L 0 5 10 15 20 2 30 Concentration of Protein ug mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit protein assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in the calibration This plot shows the line corresponding to the curve fitting algori
6. in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Limited Use Label License Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee o
7. in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides Core range high confidence 1 ng mL to 500 ng mLt Qubit dsDNA HS Assay Kit 032851 100 Extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA Useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit Q32854 500 Accurate in the presence of RNA salts solvents proteins and free nucleotides Core range high confidence 5 ng mL to 1000 ng mLt Extended ranges moderate confidence 1 ng mL to 5 ng mL and 1000 ng mL to 1200 ng mLt QUD IE SSDNA Assay Kit Q10212 109 SONA Useful for quantitation of oligos primers denatured DNA PCR products Accurate in the presence of salts urea solvents proteins ATP and agarose Core range high confidence 25 ng mL to 500 ng mLt Qubit RNA Assay Kit Q32852 100 Extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1000 ng mLt RNA Useful for quantitation of samples for microarray RT PCR and Northern blot procedures Accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 RNA Core range high confidence 0 1 g mL to 5 ug mLt Extended ranges moderate confidence 0 05 ug mL to 0 1 g mL and 5 6 ug mLt U
8. invitrogen MOLECULAR PROBES Qubit Protein Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Room temperature e Qubit Prot liv reagent 300 uL or 1 5 mL 200X concentrate in 1 2 propanediol e Desiccate Component A Protect from light m Qubit protein buffer 60 mL or 300 mL Not applicable e Room temperature Component B 5 Qubit protein standard 1 1mLor5mL 0 ng uL in TE buffer with 2 mM sodium azide Component C ce Qubit proteiin standard 2 1mLor5mL 200 ng uL in TE buffer with 2 mM sodium azide lt 4 C Component D pm Dole protein standard 3 1mLor5mL 400 ng uL in TE buffer with 2 mM sodium azide Component E When stored as directed kits are stable for 6 months Introduction The Qubit Protein Assay Kits for use with the Qubit 2 0 Fluorometer make protein quantitation easy and accurate The kits provide concentrated assay reagent dilution buffer and pre diluted BSA standards Simply dilute the reagent using the buffer provided add your sample any volume from 1 20 uL is acceptable then read the concentration using the Qubit 2 0 Fluorometer The assay is accurate for initial sample concentrations from 12 5 ug mL to 5 mg mL and exhibits low protein to protein variation Appendix Figure 1 The assay is performed at room temperature and the signal is stable for 3
9. it 2 0 Fluorometer gives values for the Qubit protein assay in ug mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 2 X where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer that is if the Qubit 2 0 Fluorometer gave a concentration in g mL the result of the equation will be in ug mL Qubit Protein Assay Kits 4 Dilution Calculator Appendix Protein to protein variation of the Qubit Protein Assay 2 1 2 2 2 3 2 4 2 5 2 6 After the sample measurement is completed press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window opens showing the current unit selection indicated by a red dash Select the unit for your original sample concentration by touching the desi
10. maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches SDS Safety Data Sheets SDSs are available at www lifetechnologies com sds Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the product packaging tube pouch or box Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored
11. nd insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation time To allow the Qubit protein assay to reach optimal fluorescence incubate the tubes for 15 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature For greatest accuracy of the protein assay the incubation time of the samples should be within 10 minutes of the incubation time of the standards Photobleaching of the Qubit reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature Appendix Figure 2 Note that the temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single
12. r other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 2011 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit Protein Assay Kits 9
13. red unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units for example if you change the unit to pg uL the button displays pg uL To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved as a csv file and tagged with a time and date stamp To exit the Dilution Calculator screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values BSA Ovalbumin Lysozyme B Casein Myoglobin a Casein Pepsin IgG Histone ADRS O OEMS Fluorescence 0 0 1 0 2 0 3 0 4 0 5 0 Protein ug Figure 1 Low protein to protein variation in the Qubit protein assay Q33211 Q33212 Solutions of the following proteins were prepared diluted and assayed in the Qubit protein assay bovine serum albumin BSA chicken eg
14. seful for quantitation of samples for microarray RT PCR and Northern blot procedures Accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein Assay Kit Q33211 100 Qubit Protein Assay Kit Q33212 500 Protein Core range high confidence 1 25 g mL to 25 ug mLt Extended ranges moderate confidence 1 ug mL to 1 25 ug mL and 25 g mL to 26 ug mLt Little protein to protein difference in signal Accurate in the presence of DTT B mercaptoethanol amino acids and DNA Signal is stable for 3 hours Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit Protein Assay Kits 8 Product List current prices may be obtained from our website or from our Customer Service Department Cat no Product name Unit size Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer aie 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 g for use with the Qubit 2 0 Fluorometer 0 cece c cece eee cence nee en eens 1 kit Related products Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 02 cece cee eee cence nee ee eens 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cece cece cece cence nee ee eens 1 ki
15. sesrerirrrerssiisedtends d rinkis Er eee eee Eain a E AE each Contact Information Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Fax 1 760 602 6500 Email techsupport lifetech com European Headquarters Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Phone 44 141 814 6100 Toll Free Phone 0800 269 210 Toll Free Tech 0800 838 380 Fax 44 141 814 6260 Tech Fax 44 141 8146117 Email euroinfo invitrogen com Email Tech eurotech invitrogen com Japanese Headquarters LOOP X Bldg 6F 3 9 15 Kaigan Minato ku Tokyo 108 0022 Japan Phone 81 3 5730 6509 Fax 81 3 5730 6519 Email jpinfo invitrogen com Additional international offices are listed at www lifetechnologies com These high quality reagents and materials must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Read the Safety Data Sheet provided for each product other regulatory considerations may apply Obtaining Support For the latest services and support information for all locations go to www lifetechnologies com At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support techsupport lifetech com Search for user documents SDSs vector
16. t 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 12 and 2 1 2 6 can be found in the user manual accompanying the instrument Set up the required number of 0 5 mL tubes for standards and samples The Qubit protein assay requires 3 standards Note Use only thin wall clear 0 5 mL optical grade real time PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 Label the tube lids Note It is important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Make the Qubit working solution by diluting the Qubit protein reagent 1 200 in Qubit protein buffer Use a clean plastic tube each time you make the Qubit working solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube requires 190 uL of Qubit working solution and each sample tube requires anywhere from 180 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 7 samples prepare enough working solution for the samples and 3 standards
17. t Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cee cece nec e cence tenn eee eeee 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee eee e tenn ene eee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 0 ccc e cece cence een een eens 1 kit Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00 cece eee eee ene eee eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0c cece eee cence nee ee eens 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 06 cece eee eee e ene eee eeee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee ee ee een eee eens 1 kit Q32856 Qubit assay tubes set 0f 500 iccsesavicnsaigvarnciascisneseaumaduetetens eevee AAA AAEE E EAEE ERE AAAS 1 set Q32866 Qubit 20 FIUOrOMEt e rrrsissseni rissies r sr EEEE PEPETA DURS sdb EEEE TEEPE OREERT EEES EEEE EES Seed EINE Ga KENTE each Q32867 Q bit 20 Fl urometer USB ncoaiinccad canoes Sega a GE enemies dena E E EA A E E Sean ERSE adnate each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement s s i
18. thm a modified Hill plot used in the calculation of concentration data for the Qubit protein assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Qubit Protein Assay Kits 6 Contaminants tolerated by the Qubit Protein Assay Table 2 Effect of contaminants in the Qubit protein assay tested over the range of 1 25 g mL to 25 ug mL Final concentration Concentration in Concentration in Contaminant in the assay 20 pL sample 10 uL sample Result Sodium chloride 20 mM 200 mM 400 mM OKt Magnesium chloride 2mM 20 mM 40mM OK Potassium chloride 200 mM 200 mM 400 mM OK Calcium chloride 2mM 20 mM 40mM OK Ammonium sulfate 5mM 50 mM 100 mM OKt DTT 1mM 10mM 20 mM OKt 6 Mercaptoethanol 1mM 10 mM 20 mM OK EDTA 1mM 10 mM 20 mM OK Sodium azide 1mM 10 mM 20 mM OK HEPES pH 7 4 5mM 50mM 100 mM OK E g phosphate 5 mM 50 mM 100 mM OK ene pH 7 4 kis hwa SONNE modiestion required Sucrose 50 mM 500 mM 1M OK Sucrose 100 mM 1M 2M NR Glycerol 1 10 20 OKt Imidazole 1 25 mM 12 5mM 25 mM OK SDS 0 01 0 1 0 2 OKt SDS 0 02 0 2 0 4 NR Tween 20 0 001 0 01 0 02 NR Triton X 100 0 001 0 01 0 02 NR Amino acids 100 g mL 1 mg mL 2 mg mL OK dNTPs 100 uM 1mM 2mM OKt DNA 5 g mL 50 ug mL 1
19. tube you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit Protein Assay Kits 2 Handling and disposal Experimental Protocol 1 1 1 2 1 3 1 4 1 5 1 6 1 7 No data are currently available addressing the mutagenicity or toxicity of the Qubit protein reagent Component A This reagent is an organic dye and is provided as a solution in 1 2 propanediol Treat the Qubit protein reagent with the same safety precautions as other materials with similar properties and dispose of the dye in accordance with local regulations The protocol below assumes that you are preparing standards for calibrating the Qubi
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