Qubit™ Protein Assay Kits - Ecologie Microbienne Lyon
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1. RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 g mL to 0 1 g mL and 5 6 ug mLt useful 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 g mL and 25 g mL to 26 ug mLt little protein to protein difference in signal accurate in the presence of DTT 8 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 ete 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cece cece cece e een e ences 1 kit Related products Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with th2. 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 Qubit proteinreagent 300 uL or 1 5 mL 200X concentrate in 1 2 propanediol Desiccate Component A Protect from light Qubit protein buffer 60 mL or 300 mL Not applicable Room temperature Component B Qubit protein standard 1 1mLor5mL 0 ng uL in TE buffer with 2 mM sodium azide Component C UDI protein standard p2 1mLor5 mL 200 ng ul in TE buffer with 2 mM sodium azide lt 4 C Component D Qubit protein standard 3 1mLor5mL 400 ng L 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 between 1 uL and 20 uL is acceptable and 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 hours C
3. 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 pg mL 1 mg mL 2 mg mL OK dNTPs 100 uM 1mM 2mM OK t DNA 5 ug mL 50 g mL 100 ug mL OK t 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 approxima 10 perturbation or as NR e in 20 uL or 10 uL sample volumes are also listed Results are given as OK usually less than not recommended t An 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 tt For 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 will help you choose a kit based on the target molecule being measured and the number of assays you require Product Cat no numberof Target Notes Assays Qubit dsDNA BR
4. Assay Kit 032850 100 core range high confidence 0 01 ug 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 e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides i 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 032854 500 e accurate in the presence of RNA salts solvents proteins and free nucleotides core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and en 1 000 ng mL to 1 200 ng mLt Qube ssDNA Assay Kit Q10212 109 SSDNA useful for quantitation of oligos primers denatured DNA PCR products e 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 1 000 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
5. EEEE EEEE SEERE TERE ea each Q32867 Oubit 20 Fluorometer USB ssrisssereresere nean tea a EERE a NAE A a sa Sees onan EE each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2 6 cece cece nen titer ie iria tenari iniinis each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must b
6. ample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit protein assay in g 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 i e 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 Upon completion of the sample measurement 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 have 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 orig
7. calibration guidelines 1 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 Insert the tube containing Standard 3 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 3 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the result will be 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 next page 1 14 To read the next sample remove the s
8. e Qubit 2 0 Fluorometer 0 cece eee eee ene eee e eee 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee n eee eens 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cece teen ene eee eeee 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece cece eee e eee cent ene e nn eeee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee eee eee n een eens 1 kit Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00sec cece eee eee eee eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee e cence nee e eens 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cence nee e eens 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 e cece e eee eee eee e eee 1 kit Q32856 Qubit assay tubes setof 500 rapis cess aaa EAE A OSEAS ESETE AE EA NEEE EAEAN 1 set Q32866 Qubit 2 0 FlUOTOMeCtEl sssr cece desis cess trisa rE EEEE saad E EDAD FEESSIE SETETE EEEIEE EREE AEE EEEE
9. e used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in
10. ein 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 egg 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 T 160 4409 w 140 4 g o 22 120 sE 22 100 4 GE 8 80 TO Q 60 g oN 40 co E 20 e amp 0 r r r r 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs 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 oO L 0 5 10 15 20 25 30 Concentration of Protein ug mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit protein as
11. erature and 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 because 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 see Appendix Figure 2 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 reading
12. ies Corporation All rights reserved This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit Protein Assay Kits 9
13. inal sample concentration is displayed press ng mL A pop up window showing the current unit selection indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired 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 e g if you change the unit to pg L the button will display pg L 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 LEE OEMS Fluorescence 0 0 1 0 2 0 3 0 4 0 5 0 Protein ug Figure 1 Low prot
14. manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technolog
15. ommon 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 although 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 1 October 2010 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 is 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 temp
16. s of a single 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 in 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 1 8 No data are 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 you are preparing standards for calibra
17. say The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm 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 1 25 ug mL to 25 ug mL Contaminant ee in concentration an Concentrationin Result TORY 20 pL Sample 10 pL Sample Sodium chloride 20 mM 200 mM 400 mM OK t Magnesium chloride 2mM 20mM 40 mM OK Potassium chloride 200 mM 200 mM 400 mM OK Calcium chloride 2mM 20 mM 40 mM OK Ammonium sulfate 5mM 50 mM 100 mM OK t DTT 1 mM 10 mM 20 mM OK t B Mercaptoethanol 1 mM 10 mM 20 mM OK EDTA 1 mM 10 mM 20 mM OK Sodium azide 1 mM 10 mM 20 mM OK HEPES pH 7 4 5 mM 50 mM 100 mM OK oe phosphate 5mM 50 mM 100 mM OK rsp sues om Stns Mode required Sucrose 50 mM 500 mM 1M OK Sucrose 100 mM 1M 2M NR Glycerol 1 10 20 OK t Imidazole 1 25mM 12 5 mM 25 mM OK SDS 0 01 0 1 0 2 OK t SDS
18. ting the Qubit 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 15 and 2 1 2 6 can be found in the user manual accompanying the instrument Set up the number of 0 5 mL tubes you need 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 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 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 uL to 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 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the t
19. ubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being 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 It is also 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 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 between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow all tubes to incubate at room temperature for 15 minutes Qubit Protein Assay Kits 3 1 9 On the Home Screen of the Qubit 2 0 Fluorometer press Protein The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for
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