Protein Quantification Assay - MACHEREY
Contents
1. Type of assay Required Protein amount Determinable sample volume per assay protein concentration Microplate 20 uL 1 60 pL 0 6 20 ug 30 1000 ng uL Semi micro cuvette 200 uL 10 600 uL 6 200 ug 30 1000 ng uL Micro cuvette 40 uL 1 120 uL 1 2 40 ug 30 1000 ng uL Low volume 7 5 ub 0 47 7 5 ug 60 1000 ng uL 740967 50 740967 250 Type of Protein Calibration Total Protein Calibration Total assay deter curves number of determi curves number of mina 7 points reactions nation 7 points reactions tion per curve per curve Microplate 50 6 Approx 100 250 25 Approx 450 Semi micro cuvette 3 1 Approx 10 26 3 Approx 50 Micro cuvette 35 3 Approx 55 130 15 Approx 235 Low volume 800 25 Approx 1000 3000 70 Approx 3500 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 2 3 Handling preparation and storage of starting materials After dissolving protein in Protein Solving Buffer PSB or Protein Loading Buffer PLB with or without Reducing Agent TCEP Laemmli buffer or analogs freeze your protein samples for long term storage or keep samples at 4 C for short term storage Before use make sure that the samples are free of precipitates If necessary heat to approximately 30 C in order to dissolve any possible SDS precipitate Subsequently spin sample briefly to remove any further insoluble matter Protein samples obtained with NucleoSpin RNA Protein or N2. 0 01 0 33 ug uL 60 uL 0 6 20 ug 0 03 1 0 ug uL 20 uL 0 6 20 ug 0 6 20 ug uL 1 uL 0 6 20 ug Table 3 Semi micro cuvette assay Recommended sample volumes for protein quantification Recommended Protein amount sample volume Expected protein concentration 0 01 0 33 ug uL 600 uL 6 200 ug 0 03 1 0 ug uL 200 uL 6 200 ug 0 6 20 ug uL 10 uL 6 200 ug Table 4 Microcuvette assay Recommended sample volumes for protein quantification Recommended sample volume Expected protein concentration Protein amount per microcuvette 0 01 0 33 ug uL 120 uL 1 2 40 ug 0 03 1 0 yg uL 40 uL 1 2 40 yg 1 2 40 yg uL Tub 1 2 40 ug Table 5 Low volume assay Recommended sample volumes for protein quantification Expected protein concentration Recommended sample volume Protein amount per microcuvette 0 06 1 ug uL 7 5 uL 0 47 7 5 ug 10 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 2 6 Alternative wavelengths for extinction measurement A wavelength in the range of 530 700 nm is recommended for light extinction measurements Figure 2 shows the dependency of correlation coefficient on the wavelength used for light extinction measurement 0 8 0 6 5 0 4 0 2 4 Correlation coefficient 0 0 400 500 600 700 800 Wave length nm Figure 2 Dependency of correlation coefficient on the
3. For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com IN The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin 14 MACHEREY NAGEL 05 2014 Rev 04 Microplate assay 5 5 1 Protocols Microplate assay procedure Before starting the preparation Check if the BSA reference protein stock solution was prepared according to section 3 Make sure that there are no precipitates in Protein Solving Buffer PSB and in the reverence protein BSA solution if necessary heat to approx 30 C 1 Prepare a BSA reference protein dilution series Number seven reaction tubes according to column A see table below 1 BSA stock solution Add 50 uL Protein Solving Buffer PSB to tubes 2 7 column B Add BSA solution to tubes 2 6 according to column C The resulting protein concentration and amount are shown in columns D and E PSB contains detergent When pipetting BSA and PSB solutions avoid bubble formation and foaming as far as possible A B Cc D E Tube Add PSB Add BSA solution Resulting BSA Resulting BSA to tube to tube concentration in 20 uL 1 BSA stock solution 1 ug L 20 ug 2 50 uL 50 uL from tube 1 0 5 ug uL 10 ug 3 50 uL 50 uL from tu
4. Protein Quantification Assay User manual May 2014 Rev 04 MACHEREY NAGEL www mn net com Protein Quantification Assay Protocol at a glance Rev 04 Microplate assay Semi micro Micro cuvette Low volume cuvette assay assay assay Prepare BSA Dispense Dispense Dispense Dispense reference pro 50 pL PSB 250 pL PSB 50 pL PSB 20 pL PSB tein dilution per tube 2 7 per tube 2 7 per tube 2 7 per tube 2 6 series Pipette 50 pL BSA stock solution into tube 2 then 50 uL from 2 into 3 etc Pipette 250 uL BSA stock solution into tube 2 then 250 uL from 2 into 3 etc Pipette 50 pL BSA stock solution into tube 2 then 50 uL from 2 into 3 etc Pipette 20 pL BSA stock solution into tube 2 then 20 uL from 2 into 3 etc Dilution series sufficient for Two calibration curves One calibration curve One calibration curve Two calibration curves Dispense dilution series 20E 200 HE On Zah Dispense your 20 uL 200 uL 40 uL 75 uL protein sample 1 60 uL 10 600 uL 1 120 uL gt M Fill up dilution series and 40 uL 400 uL 80 uL _ sample with final vol 60 uL final vol 600 uL final vol 120 uL PSB Add Quantification 40 uL 400 uL 80 uL 5 ul Reagent QR 6 Incubate 30 min at room temperature 7 Measure light extinction At 570 nm 530 nm 700 nm 8 Calculate protein concentration Make sure that the signal of your sample lies wit
5. A method for protein assay in Laemmli buffer Analytical Biochemistry 219 144 146 Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680 685 1970 Lowry OH etal 1951 Protein measurement with the Folin phenol reagent J Biol Chem 193 265 275 Smith PK etal 1985 Measurement of protein using bicinchoninic acid Anal Biochemem 150 1 76 85 6 5 Product use restriction warranty Protein Quantification Assay kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable fo
6. cell and influence on the protein quantification Molecule Content Content per Extinction signal Extinction signal per cell one million obtained with the obtained relative to cells Protein Quantification total protein Assay relative to the reverence protein BSA DNA 6pg 6 ug 50 70 3 5 RNA 10 30 pg 10 30 ug 10 40 1 12 Protein 100 200 pg 100 200 ug 100 100 Total 104 117 Signal obtained from total cell extract containing RNA and DNA relative to nucleic acid free total protein 104 117 6 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay Table 1 Kit specifications at a glance Protein Quantification Assay Sample size 1 600 uL containing 0 6 200 yg protein BSA equivalents Microplate assay 0 6 20 pg protein BSA equivalents in 20 uL corresponding to 30 1000 ng uL Semi micro cuvette assay 6 200 ug protein BSA equivalents in 200 uL corresponding to 30 1000 ng uL Sample type Protein solved in Protein Solving Buffer PSB Laemmli buffer or equivalent preferable free of nucleic acids Protein concentration Approx 30 1 000 ng uL standard range or Approx 10 20 000 ng uL extended range Correlation coefficient 0 97 1 00 Wavelength for light extinction measurement 570 nm 530 700 nm Time Approx 40 min Kit specifictions vary depending on the type of assay Please find more detailed information in the tables below
7. customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MAC
8. dilution series 1 BSA stock solution 2 6 BSA dilutions 7 BSA free PSB 18 MACHEREY NAGEL 05 2014 Rev 04 Semi micro cuvette assay Dispense your protein samples Pipette 200 uL of your samples to new microcentrifuge tubes Alternatively 10 600 uL of sample can be applied Fill up dilution series and protein samples Add 400 uL PSB to each microcentrifuge tube dilution series and protein samples Final volume is 600 uL Alternatively when applying other sample volumes than 200 uL in step 3 fill up with PSB to a final volume of 600 uL e g 100 uL sample 500 uL PSB Add Quantification Reagent QR Add 400 uL Quantification Reagent QR to each microcentrifuge tube dilution series and protein samples Shake tubes until a complete color change from blue to yellow occurs Caution Quantification Reagent QR contains hydrochloric acid Wear protective clothing and goggles Incubate Incubate microcentrifuge tubes for 30 min at room temperature Shake tubes after incubation Do not centrifuge tubes at this point A variation in incubation time may result in reduced signal and loss of sensitivity An incubation time of 30 5 min is recommended Measure light extinction Transfer the solution of each tube to a suitable semi micro cuvette Measure light extinction photometrically at 570 nm Light extinction can be measured in the range of 530 700 nm Typical correlation coefficients c
9. 1 6 into 7 5 uL of 1 5 mL microcentrifuge tubes not supplied dilution series 1 BSA stock solution 2 5 BSA dilutions 6 BSA free PSB 24 MACHEREY NAGEL 05 2014 Rev 04 Low volume assay Dispense your protein samples 7 5 pL of Pipette 7 5 uL of your samples to new microcentrifuge tubes samples Fill up dilution series and protein samples Not necessary Proceed directly with step 5 Add Quantification Reagent QR Add 5 uL Quantification Reagent QR to each tube dilution series and protein samples 5uLQR Mix e g by pipetting up and down until a complete color Mix change from blue to yellow occurs Caution Quantification Reagent QR contains hydrochloric acid Wear protective clothing and goggles Incubate Incubate tubes for 30 min at room temperature Incubate Shake tubes after incubation Do not centrifuge at this point 30 min Avariation in incubation time may result in reduced signal and loss of sensitivity An incubation time of 30 5 min is recommended Measure light extinction Transfer 10 uL of the solution of each tube to a suitable low volume photometer with 1mm path length Measure light extinction photometrically at 570 nm Avoid bubbles in the solution because they severely disturb the measurement Measure Caution The solution to be measured contains HCI check the extinction compatibility of your instrument with HCI Do not spill Immediately at 570 nm remove solution from
10. 1 E i BR semi micro cuvette 2 01 microplate w y 0 0036x F y 0 176x 2 R 0 995 f R 0 998 0 001 Hy ru ug a 0 001 a 0 1 1 10 100 1000 0 01 0 1 1 BSA amount per assay pg BSA concentration ug L Figure 1 Correlation between BSA amount and extinction signal as well as between BSA concentration and extinction signal For the microplate assay BSA was supplied in 20 uL path length for extinction measurement was 3mm For the semi micro cuvette assay BSA was supplied in 200 uL path length for extinction measurement was 10 mm 2 5 Recommended sample volumes As guidance follow the recommendations of Table 2 Table 5 to choose an appropriate volume of your sample for measuring For the initial determination of protein concentration in samples containing hard to estimate protein amounts measurement of multiple sample volumes e g 2 uL 5 uL 50 uL is recommended This will increase the probability that one of the measured protein amounts lies within the range of the calibration curve For protein samples obtained with NucleoSpin RNA Protein or NucleoSpin TriPrep see the respective user manual for a first estimation of the protein yield MACHEREY NAGEL 05 2014 Rev 04 9 Protein Quantification Assay Table 2 Microplate assay Recommended sample volumes for protein quantification Recommended sample volume Expected protein concentration Protein amount per well
11. 21 969 270 tech bio mn net com Trademarks NanoPhotometer is a trademark of IMPLEN Ltd NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 34 MACHEREY NAGEL 05 2014 Rev 04
12. 45 0 033 0 111 2 10 0 130 0 042 0 166 0 088 0 040 0 354 0 031 0 132 3 5 0 072 0 037 0 199 0 111 0 046 0 330 0 032 0 250 4 2 5 0 050 5 1 25 0 043 6 0 625 0 039 7 0 0 032 blank Correlation 0 998 coefficient MACHEREY NAGEL 05 2014 Rev 04 27 2 The correct raw data is obtained by subtracting the blank value from the values of the protein standards and unknown samples BSA Reference US US US US US US US amount protein A B Cc D E F G per well dilution ng series 1 20 0 213 0 008 0 179 0 068 0 013 0 313 0 001 0 079 2 10 0 098 0 011 0 134 0 056 0 008 0 322 0 002 0 100 3 5 0 040 0 005 0 167 0 079 0 014 0 298 0 001 0 218 4 2 5 0 018 5 1 25 0 011 6 0 625 0 007 7 0 0 blank 3 Create a standard curve by plotting the extinction values versus the reference protein amount per well Plot a linear regression for the set of standards and calculate the equation of this line Calibration curve blank corrected 0 250 y 0 0107x 0 0052 0 200 4 R 0 995 0 150 0 100 Extinction E579 nm 0 050 0 000 In this case the equation is y 0 0107x 0 0052 28 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 4 Calculate protein concentration Insert the measured extinction of each unknown sample for x amount of protein per well to calculate the
13. GEL 05 2014 Rev 04 23 Low volume assay 5 4 Low volume assay procedure Before starting the preparation Check if the BSA reference protein stock solution was prepared according to section 3 Make sure that there are no precipitates in Protein Solving Buffer PSB and in the reverence protein BSA solution if necessary heat to approx 30 C 1 Prepare a BSA reference protein dilution series Number six reaction tubes according to column A see table below 1 BSA stock solution Add 20 uL Protein Solving Buffer PSB to tubes 2 6 column B Add BSA solution to tubes 2 5 according to column C The resulting protein concentration and amount are shown in columns D and E A B C D E Tube Add PSB Add BSA solution Resulting BSA Resulting BSA to tube to tube concentration in 20 pL 1 BSA stock solution 1 ug uL 7 5 ug 2 20 uL 20 uL from tube 1 0 5 ug uL 3 75 ug 3 20 uL 20 uL from tube 2 0 25 ug uL 1 88 ug 4 20 uL 20 uL from tube 3 0 125 ug uL 0 94 ug 5 20 uL 20 uL from tube 4 0 063 ug uL 0 47 ug 6 20 uL 0 ug uL 0 ug The prepared BSA dilutions series is sufficient for the determination of two calibration curves Freeze BSA stock solution for storage Keep dilutions series at room temperature during use and dispose all dilutions at the end of a working day 2 Dispense dilution series into microcentrifuge tubes Pipette 7 5 uL of each dilution series solution
14. HEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL 05 2014 Rev 04 33 Protein Quantification Assay MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24
15. L sample 110 uL PSB 5 Add Quantification Reagent QR Pipette 80 uL Quantification Reagent QR to each tube dilution series and protein samples 80 pL QR Shake tube until a complete color change from blue to yellow Shake tube occurs Caution Quantification Reagent QR contains hydrochloric acid Wear protective clothing and goggles 6 Incubate Incubate tubes for 30 min at room temperature 7 Incubate Shake tubes after incubation Do not centrifuge tubes at this 30 min point A variation in incubation time may result in reduced signal and loss of sensitivity An incubation time of 30 5 min is recommended 7 Measure light extinction Transfer the solution of each tube to a suitable microcuvette Measure light extinction photometrically at 570 nm Measure extinction Light extinction can be measured in the range of 530 700 nm at 570 nm Typical correlation coefficients concentration of BSA versus extinction value of 0 97 1 00 are obtained within this wavelength range 22 MACHEREY NAGEL 05 2014 Rev 04 Microcuvette assay Calculate protein concentration Calculate protein concentration of samples in relation to the BSA dilution series Calculate protein Make sure that the protein concentration of your sample lies within concentration the range of the largest 1 and the smallest 6 concentration of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range MACHEREY NA
16. be 2 0 25 ug uL 5 ug 4 50 uL 50 uL from tube 3 0 125 ug uL 2 5 ug 5 50 uL 50 uL from tube 4 0 063 ug uL 1 25 ug 6 50 uL 50 uL from tube 5 0 031 ug L 0 625 ug 7 50 uL 0 ug uL 0 ug The prepared BSA dilutions series is sufficient for the determination of two calibration curves Freeze BSA stock solution for storage Keep dilutions series at room temperature during use and dispose all dilutions at the end of a working day MACHEREY NAGEL 05 2014 Rev 04 15 Microplate assay 2 Dispense dilution series into microplate Add 20 yL of each dilution series solution 1 7 into 20 uL of microplate wells dilution series 1 BSA stock solution 2 6 BSA dilutions 7 BSA free PSB 3 Dispense your protein samples 20 uL of Pipette 20 uL of your samples to empty wells samples Alternatively 1 60 uL of sample can be applied 4 Fill up dilution series and protein samples Add 40 uLPSB to each well dilution series and protein samples Final volume is 60 uL 40 pL PSB Alternatively when applying other sample volumes than 20 uL in step 3 fill up with PSB to a final volume of 60 uL e g 10 uL sample 50 uL PSB 5 Add Quantification Reagent QR Add 40 uL Quantification Reagent QR to each well dilution series and protein samples 40 uL QR Shake microplate until a complete color change from blue to Shake yellow occurs microplate Caution Quantification Reagent QR contains hydrochloric ac
17. c D E Tube Add PSB Add BSAsolution Resulting BSA Resulting BSA to tube to tube concentration in 20 pL 1 BSA stock solution 1 ug uL 20 ug 2 50 uL 50 uL from tube 1 0 5 ug uL 10 ug 3 50 uL 50 uL from tube 2 0 25 ug uL 5 ug 4 50 uL 50 uL from tube 3 0 125 ug uL 2 5 ug 5 50 uL 50 uL from tube 4 0 063 ug uL 1 25 ug 6 50 uL 50 uL from tube 5 0 031 ug uL 0 625 ug 7 50 uL 0 ug L 0 ug The prepared BSA dilutions series is sufficient for the determination of one calibration curve Freeze BSA stock solution for storage Keep dilutions series at room temperature during use and dispose all dilutions at the end of a working day Dispense dilution series into microcentrifuge tubes Pipette 40 uL of each dilution series solution 1 7 into 40 uL of 1 5 mL microcentrifuge tubes not supplied dilution series 1 BSA stock solution 2 6 BSA dilutions 7 BSA free PSB MACHEREY NAGEL 05 2014 Rev 04 21 Microcuvette assay 3 Dispense your protein samples j 40 uL of Pipette 40 uL of your samples to new microcentrifuge tubes samples Alternatively 1 120 uL of sample can be applied 4 Fill up dilution series and protein samples Add 80 uL PSB to each well dilution series and protein samples Final volume is 120 uL 80 pL PSB Alternatively when applying other sample volumes than 40 uL in step 3 fill up with PSB to a final volume of 120 uL e g 10 u
18. ccording to the semi micro cuvette assay three protein determinations plus seven calibration points The kit REF 740967 250 is sufficient for 250 protein determinations plus 25 calibration curves with seven calibration points each approx 450 reactions in total according to the microplate assay Alternatively the kit is sufficient for approx 50 reactions according to the semi micro cuvette assay 26 protein determinations plus three calibration curves with seven calibration points each Following the microplate assay procedure the kit allows the determination of protein amount exemplary BSA in the range of 0 6 20 ug per assay provided in a standard volume of 20 uL Protein Solving Buffer PSB alternatively 1 60 uL This corresponds to a protein concentration of 30 1000 ng uL This concentration range can be expanded to 10 20 000 ng uL if alternative sample volumes 1 60 uL are applied Following the semi micro cuvette assay procedure the kit allows the determination of protein exemplary BSA amount in the range of 6 200 ug per assay provided in a standard volume of 200 uL Protein Solving Buffer PSB This corresponds to a protein concentration of 30 1000 ng uL One microgram DNA causes ca 50 70 of the extinction signal caused by one microgram protein BSA One microgram RNA causes ca 10 40 of the extinction signal caused by one microgram protein BSA DNA RNA and protein content of a typical
19. ck solution was prepared according to section 3 Make sure that there are no precipitates in Protein Solving Buffer PSB and in the reverence protein BSA solution if necessary heat to approx 30 C Prepare a BSA reference protein dilution series Number seven reaction tubes according to column A see table below 1 BSA stock solution Add 250 uL Protein Solving Buffer PSB to tubes 2 7 column B Add BSA solution to tubes 2 6 according to column C The resulting protein concentration and amount are shown in columns D and E A B C D E Tube Add PSB Add BSA solution Resulting BSA Resulting BSA to tube to tube concentration in 20 uL 1 BSA stock solution 1 ug uL 200 ug 2 250 uL 250 uL from tube 1 0 5 ug uL 100 ug 3 250 uL 250 uL from tube 2 0 25 ug uL 50 ug 4 250 uL 250 uL from tube 3 0 125 ug uL 25 ug 5 250 uL 250 uL from tube 4 0 063 ug uL 12 5 ug 6 250 uL 250 uL from tube 5 0 031 ug uL 6 25 ug 7 250 uL 0 ug L 0 ug The prepared BSA dilutions series is sufficient for the determination of one calibration curve Freeze BSA stock solution for storage Keep dilutions series at room temperature during use and dispose all dilutions at the end of a working day 2 Dispense dilution series into microcentrifuge tubes Pipette 200 uL of each dilution series solution 1 7 into 200 pL of 1 5 mL microcentrifuge tubes not supplied
20. ent QR 20 mL 20 mL User Manual 1 1 1 2 Consumables and equipment to be supplied by user Consumables Microplates flat bottom e g UV Star Microtiter plate 96 well F bottom Greiner bio one REF 655801 similar non UV transparent microtiter plates are also suitable or semi micro cuvettes e g Plastibrand 1 5 mL semi micro disposable cuvettes Brand REF 759115 or micro cuvettes e g Plastibrand UV Cuvette micro Brand REF 759220 1 5 mL microcentrifuge tubes to prepare dilution series for the calibration curve and to set up reactions when following the semi micro cuvette assay procedure Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes to clean microcentrifuge lids if necessary Vortex mixer Mixer or shaker for microplates Photometer set to 570 nm 570 nm is recommended other wavelength settings in the range of 530 700 nm are also suitable either for microplates microplate assay procedure for semi micro microcuvettes semi micro cuvette and micro cuvette assay procedure or for low volume analysis e g NanoDrop Thermo Scientific NanoVue GE Healthcare or NanoPhotometer Implen Personal protection equipment e g lab coat gloves goggles For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 2 Product description 2 1 The basic principle The Pro
21. g components of the Protein Quantification Assay contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze QR Hydrochloric acid Warning 290 319 234 261 271 280 10 25 335 302 352 304 340 Salzs ure 10 25 Achtung 305 351 338 312 332 313 337 313 390 403 233 405 406 Hazard phrases H 290 May be corosive to metals Kann gegen ber Metall korrosiv sein H 315 Causes skin irritation Verursacht Hautreizungen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 335 May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 234 Keep only in original container Nur im Originalbeh lter aufbewahren P 261 Avoid breathing dust Einatmen von Staub vermeiden P 271 Use only outdoors or in a well ventilated area Nur im Freien oder in gut bel fteten R umen verwenden P 280 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 302 352 IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen P 304 340 IF INHALED If b
22. hin the range of the calibration curve Keep tube 7 as BLANK Do not add 50 uL from tube 6 into tube 7 MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Protein Quantification Assay Table of contents 1 Components 1 1 Kit contents 1 2 Consumables and equipment to be supplied by user 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Handling preparation and storage of starting materials 8 2 4 Calibration curves 9 2 5 Recommended sample volumes 9 2 6 Alternative wavelengths for extinction measurement 11 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 13 5 Protocols 15 5 1 Microplate assay procedure 15 5 2 Semi microcuvette assay procedure 18 5 3 Microcuvette assay procedure 21 5 4 Low volume assay procedure 24 6 Appendix 27 6 1 Guidance for data analysis calculation of protein concentration 27 6 2 Troubleshooting 30 6 3 Ordering information 31 6 4 References 32 6 5 Product use restriction warranty 32 MACHEREY NAGEL 05 2014 Rev 04 3 Protein Quantification Assay 1 1 1 Components Kit contents Protein Quantification Assay 50 assays 250 assays REF 740967 50 740967 250 Protein Solving Buffer PSB 7 5 mL 40 mL BSA Bovine Serum Albumin oe img 2ximg reference protein Quantification Reag
23. id Wear protective clothing and goggles 6 Incubate Incubate microplate for 30 min at room temperature Gently shake microplate after incubation but avoid bubble Incubate formation and foaming For optimal measurement the solution 30 min surface in the microplate well should be free of bubbles and foam Light scattering caused by foam has impact on the measurement Avariation in incubation time may result in reduced signal and loss of sensitivity An incubation time of 30 5 min is recommended 16 MACHEREY NAGEL 05 2014 Rev 04 Microplate assay Measure light extinction Measure light extinction photometrically at 570 nm Light extinction can be measured in the range of 530 700 nm Typical correlation coefficients concentration of BSA versus extinction value of 0 97 1 00 are obtained within this wavelength range Calculate protein concentration Calculate protein concentration of samples in relation to the BSA dilution series Make sure that the protein concentration of your sample lies within the range of the largest 1 and the smallest 6 concentration of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range Measure extinction at 570 nm Calculate protein concentration MACHEREY NAGEL 05 2014 Rev 04 17 Semi micro cuvette assay 5 2 Semi microcuvette assay procedure Before starting the preparation Check if the BSA reference protein sto
24. ing caused by foam has impact on turbidity measrurements 30 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay Problem Possible cause and suggestions Protein Solving Buffer PSB appears turbid Low storage temperature Warm PSB to approx 30 C Similar extinction for all dilution series samples Fill level of semi micro or microcuvette not compatible with photometer Make sure that the sample volume in the semi micro cuvette is high enough to let the light beam pass through the solution Consult your photometer user manual Check the compatibility of disposable cuvettes used with your photometer consider light beam center height and cuvette fill volume 6 3 Ordering information Product REF Number of assays or preparations Protein Quantification Assay 740967 50 250 50 250 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin TriPrep 740966 10 50 250 10 50 250 Porablot transfer membranes see www mn net com bioanalysis Blotting paper see www mn net com bioanalysis DISTRIBUTION AND USE OF NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 05 2014 Rev 04 31 Protein Quantification Assay 6 4 References Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Anal Biochem 72 248 254 Karlsson JO et al 1994
25. oncentration of BSA versus extinction value of 0 97 1 00 are obtained within this wavelength range 200 uL of samples 400 pL PSB 400 pL QR Shake tubes Incubate 30 min Measure extinction at 570 nm MACHEREY NAGEL 05 2014 Rev 04 19 Semi micro cuvette assay Calculate protein concentration Calculate protein concentration of samples in relation to the BSA dilution series Make sure that the protein concentration of your sample lies within the range of the largest 1 and the smallest 6 concentration of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range Calculate protein concentration 20 MACHEREY NAGEL 05 2014 Rev 04 Microcuvette assay 5 3 Microcuvette assay procedure Before starting the preparation Check if the BSA reference protein stock solution was prepared according to section 3 Make sure that there are no precipitates in Protein Solving Buffer PSB and in the reverence protein BSA solution if necessary heat to approx 30 C Prepare a BSA reference protein dilution series Number seven reaction tubes according to column A see table below 1 BSA stock solution Add 50 uL Protein Solving Buffer PSB to tubes 2 7 column B Add BSA solution to tubes 2 6 according to column C The resulting protein concentration and amount are shown in columns D and E A B C
26. protein amount of your unknown sample y ax tb x y b a a 0 0107 slope b 0 0052 axis intercept y extinction value blank corrected x protein amount in well ug Calculation example Value from unknown sample A 0 008 0 008 0 0107 x 0 0052 x 0 008 0 0052 0 0107 x 1 2 ug Calculated protein amount per well ug BSA Reference US US US US US US US amount protein A B c D E F G per well dilution ug series 1 20 20 1 2 17 7 1 7 30 0 5 8 2 10 9 1 5 13 6 1 2 31 0 4 10 3 5 4 0 9 16 8 1 8 28 0 4 21 4 2 5 2 Mean value 5 1 25 0 9 1 2 15 7 1 6 30 0 4 13 6 0 625 0 5 7 0 0 blank If 20 uL from each protein sample was pipetted into each well the protein concentration within this 20 uL sample is calculated by Mean value 20 uL protein concentration ug uL US US US US US US US A B c D E F G Mean value protein amount ug 1 2 15 7 1 6 30 0 4 13 Protein concentration ug L 0 06 0 75 0 35 0 08 1 5 0 02 0 65 MACHEREY NAGEL 05 2014 Rev 04 29 Protein Quantification Assay 5 Interpretate the results Results within the range of the reference dilution series are trustworthy Results higher than for the most concentrated reference dilution should be considered with care Do not extrapolate just interpolate Remeasure your sample with a smaller aliquot Results smaller than fo
27. r IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or 32 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the
28. r the most diluted reference protein sample should be interpreted with care Remeasure the sample using a larger aliquot 6 2 Troubleshooting Problem Possible cause and suggestions Lowest value of calibration curve cannot be measured Storage of dilution series Do not store dilution series of the BSA reference protein Prepare fresh dilution series Photometer microplates or cuvettes Sensitivity of the assay may be influenced by the type of photometer microplates or cuvettes used If the lowest calibration point is not discriminated against background prepare a calibration series with higher BSA amounts Samples appear turbid after addition of Quantification Reagent QR High protein concentration As long as the measured extinction of your sample falls within the range of the calibration curve this is acceptable Varying results upon multiple measurements Samples not mixed immediately before extinction measurement Shake microplate immediately before extinction measure ment Shake reaction tubes after incubation and before transfer to semi micro cuvettes After transfer of samples to semi micro cuvettes measure extinction immediately Strictly keep to the recommended incubation time Do not centrifuge at any time after addition of Quantification Reagent QR Avoid bubble formation and foaming especially for protocol section 5 1 microplate assay procedure Light scatter
29. reathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert P 305 351 338 IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing MACHEREY NAGEL 05 2014 Rev 04 13 Protein Quantification Assay P 312 P 332 313 P 337 313 P 390 P 403 233 P 405 P 406 BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len Call a POISON CENTER doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen If skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Absorb spillage to prevent material damage Versch ttete Mengen aufnehmen um Materialsch den zu vermeiden Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort augbewahren Store locked up Unter Verschluss aufbewahren Store in corrosive resistant container with a resistant inner liner In korrosionsbest ndigem Beh lter mit korrosionsbest ndiger Auskleidung aufbewahren
30. tein Quantification Assay is a convenient and reliable kit for the determination of protein concentration in samples typically used for SDS PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis It is mainly designed for proteins solved in Protein Solving Buffer PSB or Protein Loading Buffer PLB components of NucleoSpin RNA Protein and NucleoSpin TriPrep but will also work with proteins solved in buffer as described by Laemmli 1970 or similar These protein sample buffers usually contain SDS a reducing agent dye and a component to increase the buffer density The majority of protein quantification assays are either influenced by or incompatible with SDS reducing agents or dyes commonly present in protein sample buffers The Protein Quantification Assay however is well suited for such buffer systems It is a fast and sensitive assay based on a modification of a protocol described by Karlsson et al 1994 The samples are mixed with Protein Solving Buffer PSB and subsequently incubated for 30 minutes with Quantification Reagent QR After incubation light extinction is measured photometrically Light extinction is caused by turbidity appearing after addition of Quantification Reagent QR The protein concentration is determined in reference to a BSA Bovine Serum Albumin calibration curve BSA is provided with the Protein Quantification Assay 2 2 Kit specifications Protein Quantification Assay allows the determination of pro
31. tein concentration in samples containing up to 10 SDS and comprising reducing agent e g B mercaptoethanol BME dithiothreitol DTT dithioerythritol DTE or tris 2 carboxyethyl phosphine hydrochloride TCEP buffering salts e g TRIS or BIS TRIS dye bromphenol blue and a component to create a high density of the solution e g glycerol or sucrose Protein Quantification Assay is designed for the determination of protein concentration in samples with low nucleic acid concentration as obtained with NucleoSpin RNA Protein or NucleoSpin TriPrep For samples rich in nucleic acids the quantification is less accurate For example Coomassie Brilliant Blue G 250 Bradford 1979 copper tartrate solution and Folin reagent Lowry et al 1951 Cu Cu BCA interaction Smith et al 1985 MACHEREY NAGEL 05 2014 Rev 04 5 Protein Quantification Assay Protein Quantification Assay is suited for samples comprising protein solved in buffers commonly used for SDS PAGE e g Laemmli buffer Accuracy depends on nucleic acid content of the sample For typical cultured cells e g HeLa accuracy is affected by approximatively 5 20 due to nucleic acid content The kit REF 740967 50 is sufficient for 50 protein determinations plus six calibration curves with seven calibration points each approx 100 reactions in total according to the microplate assay Alternatively the kit is sufficient for approx 10 reactions a
32. the photometer after measurement Light extinction can be measured in the range of 530 700 nm Typical correlation coefficients concentration of BSA versus extinction value of 0 97 1 00 are obtained within this wavelength range MACHEREY NAGEL 05 2014 Rev 04 25 Low volume assay Calculate protein concentration Calculate protein concentration of samples in relation to the BSA dilution series Make sure that the protein concentration of your sample lies within the range of the largest 1 and the smallest 5 concentration of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range Calculate protein concentration 26 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 6 Appendix 6 1 Guidance for data analysis calculation of protein concentration For calculation of protein concentration of unknown samples it is necessary to prepare a BSA reference protein dilution series that is generated from known protein concentrations As guidance for calculation please follow each calculation steps listed below as an example 1 Measure the extinction of the reference protein dilution series and your unknown samples US BSA Absorption US US US US US US US amount of A B Cc D E F G per well reference ng protein dilution series 1 20 0 245 0 040 0 211 0 100 0 045 0 3
33. ucleoSpin TriPrep and dissolved in Protein Solving Buffer PSB or Protein Loading Buffer PLB with or without Reducing Agent TCEP are optimal for determination of protein concentration with the Protein Quantification Assay Quantification of protein samples obtained by boiling cells or tissue directly in either PSB or PLB with or without Reducing Agent TCEP Laemmli buffer or analogs is possible but the measurement may be less accurate due to the presence of nucleic acids which interfere with the assay The extent of interference depends on the content of protein and nucleic acid in the sample Many samples like for example cultured HeLa cells or liver tissue contain much more protein than nucleic acid and thus nucleic acids cause only small interference see footnote page 7 Wear gloves at all times during the handling to reduce risk of sample contamination with skin keratins 8 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 2 4 Calibration curves Reference protein BSA dilution series give good correlations with measured light extinction Typical correlation coefficients of 0 97 1 00 are obtained in the range of approx 0 03 1 ug uL BSA concentration BSA concentration versus extinction and BSA amount versus extinction are shown in Figure 1 1 microplate E semi micro cuvette E y 0 0088x y 0 712x c 2_ c 2 g 01 RT 0 998 g 014 R 0 995 wW W E S c 2 Ss 2 0 0
34. wavelength used for extinction measurement Light extinction of BSA samples in the range of 0 3 20 ug was measured for wavelength between 400 nm and 800 nm The correlation coefficient was calculated from the BSA amount per assay 0 3 20 ug per assay and corresponding extinction signal MACHEREY NAGEL 05 2014 Rev 04 11 Protein Quantification Assay 3 Storage conditions and preparation of working solutions Attention Quantification Reagent QR contains hydrochloric acid Wear gloves and goggles All kit components should be stored at room temperature 18 25 C Storage at lower temperatures may cause precipitation in the Protein Solving Buffer PSB Kit components are stable up to one year Before starting the Protein Quantification Assay prepare the following Dissolve the reference protein BSA 1 mg in 1 mL Protein Solving Buffer PSB to obtain a 1 mg mL BSA stock solution Freeze BSA stock solution for long term storage for short term storage keep solution at 4 C If necessary dissolve any precipitate by heating the reference solution approx 30 C before use BSA stock solution 1 mg mL BSA in PSB is stable at 20 C to 20 C for six months Protein Quantification Assay 50 assays 250 assays REF 740967 50 740967 250 BSA img 2x1mg reference protein add 1 mL PSB add 1 mL PSB to each vial 12 MACHEREY NAGEL 05 2014 Rev 04 Protein Quantification Assay 4 Safety instructions The followin
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