PI-30071 - Biotium
Contents
1. 20 minutes protected from light Centrifuge tubes briefly to collect any condensation from caps and vortex to mix 7 Transfer 200 uL of each standard or sample to 96 well microplate wells to read on a fluorescence microplate reader Measure fluorescence with excitation emission at 480 598 nm Note Alternatively samples can be transferred to a fluorescence cuvette and measured using a spectrofluorometer If more than 200 uL is required for measurement scale all volumes in the assay proportionally Page 1 of 3 Table 1 Preparation of BSA standards AIMLESS Bases Peete A 7 5 uL BSA std 2 mg mL 1mL 15 B 333 uL solution A 167 uL 10 C 250 uL solution B 250 uL 5 D 250 uL solution C 250 uL 25 E 200 uL solution D 300 uL 1 F 250 uL solution E 250 uL 0 5 G 250 uL solution F 250 uL 0 25 H 100 uL solution G 150 uL 0 1 l 0 mL 250 uL 0 Table 2 Assay tolerance levels for interfering substances Compound Maximum tolerable concentration final concentration in assay SDS 0 01 Triton X 100 Below 0 001 Tween 20 Below 0 001 CHAPS Below 0 001 Sodium deoxycholate DOC Below 0 001 Urea 10 mM DTT 100 mM BME 0 1 EDTA 1mM Sucrose 10 mM 0 34 Glycerol 1 PBS 0 02X NaCl 1mM CaCl 0 01 mM MgCl 0 2 mM ZnCl 0 2 mM Ammonium sulfate 1mM Sodium azide 2mM Imidazole 50 mM Tris 1mM Glycine 100 mM Am2. Bradford and Lowry and shows superior linearity and reproducibility compared to the NanoOrange protein quantitation assay Figure 2 The assay shows minimal variability between different proteins After protein samples are heated wtih the dye the fluorescence signal is stable for up to 16 hours at room temperature AccuOrange is recommended for quantitating purified protein or antibody samples The tolerance of the AccuOrange assay to salts buffers detergents and other chemicals is shown in Table 1 The AccuOrange assay has low tolerance for non ionic detergents and is not recommended for use with cell lysates containing Triton X 100 sodium deoxycholate CHAPs or other non ionic detergents The assay can tolerate up to 0 01 SDS final concentration in assay Absorption uoissiw3 350 450 550 650 750 Wavelength nm Figure 1 Absorbance and emission spectra of AccuOrange dye with BSA in 1X AccuOrange assay buffer AccuOrange Protein Assay Kit PSF006 Assay Protocol Note see the Appendix for information on using the AccuOrange Protein Quantitation Kit with the AccuLite 470 Mini Fluorometer 1 Prepare 1X AccuOrange buffer by diluting 10X buffer component 30071A 1 10 in dH O For example add 1 mL of 10X assay buffer to 9 mL dH O 1X assay buffer can be stored at room temperature 2 Immediately before use prepare AccuOrange working solution by diluting 500X AccuOrange dye component 30071B 1 500 in
3. navigate through saved data points Data points are numbered in order of measurement 4 To erase data select Erase All and Confirm 5 To return to previous screens select Return Performing a Full Calibration Curve with AccuLite The first time you perform the assay or if unexpected results are obtained you may wish to perform a full calibration curve to verify that the assay is performing properly In this case perform the 2 point calibration as described above then read the full set of standards as if they were unknown samples Plot the standard curve as described in the product protocol AccuOrange Protein Assay Kit PSF006 x 1 Select Calibrate y Main Menu Measure Calibrate Info Data y 7 Select Measure y Accu dsDNA AccuOrange Assay 3 Return Accu dsDNA AccuOrange Assay 3 Return 2 Select AccuOrange t 8 Select AccuOrange y N 13 Select Data y Accu dsDNA AccuOrange Assay 3 Return 14 Select AccuOrange y AccuOrange AccuOrange Data Blank lt it gt Blank Measure Erase All Return Return Return 3 Insert blank tube 9 Insert sample tube 15 Use arrows to scroll select Blank select Measure through data points Or 4 J Select Erase All 00010 000 Timer y Erase All lt Std gt Data Value lt lt Measure Save Return Return Confirm 4 The 10 ug mL standa
4. 1X AccuOrange buffer For example add 10 uL of 500X AccuOrange dye to 5 mL of 1X assay buffer Note You will need about 3 mL working solution for each standard curve see Table 1 and 250 uL working solution for each sample 3 Prepare unknown samples by adding up to 10 uL of sample to 250 uL AccuOrange working solution Note You may wish to perform a few different dilutions of your unknown sample See Table 2 for the assay tolerance levels for buffer components and potential contaminants Sample dilution may reduce the concentration of interfering substances to tolerable levels 4 Prepare a protein standard curve by performing serial dilutions of the BSA standard in AccuOrange working solution as shown in Table 1 Note the standards will be measured at the concentrations shown in Table 1 without further dilution 5 Heat samples and standards to 90 C 95 C for 10 minutes protected from light Samples can be heated in microcentrifuge tubes in a water bath or heat block Screw cap tubes or cap locks are recommended to prevent caps from popping open during heating Note Alternatively samples can be heated in a multiwell plate with a heat resistant seal using a thermocycler with a heated lid Scale all volumes in the assay proportionally to fit the well volume of the plate Centrifuge the plate to collect any condensation that forms on plate seal after step 6 6 Remove samples from heat and allow to cool to room temperature for
5. Biotlum Glowing Products for Science 3159 Corporate Place Hayward CA 94545 www biotium com Revised February 6 2014 Product Information AccuOrange Protein Quantitation Kit Catalog Number 30071 T 200 assays 30071 2000 assays Kit Contents 30071 T 30071 200 assays 2000 assays AccuOrange buffer 10X 5 mL 50 mL Component contains 2 mM sodium azide 30071A T 30071A 100 uL 1mL AccuOrange dye 500X in DMSO 30071B T 30071B Bovine serum albumin BSA standard 2 mg mL 100 uL 1mL 99990 T 99990 contains 2 mM sodium azide Number of assays based on 96 well format Storage and Handling Store at room temperature Do not refrigerate AccuOrange buffer Protect AccuOrange dye from light Product is stable for at least 1 year from date of receipt when stored as recommended If precipitate forms in Assay Buffer heat to 37 C and swirl to re dissolve No data are available on the safety of AccuOrange dye Handle using universal laboratory safety precautions and dispose of the dye as chemical waste Spectral Properties Ex Em 480 598 nm with BSA in 1X assay buffer Fig 1 Product Description AccuOrange Protein Quantitation Kit is a highly sensitive fluorescence based assay for quantitating purified protein samples in 96 well format The detection range of the assay is 0 1 15 ug mL protein AccuOrange is much more sensitive than traditional protein quantitation assays such as BCA
6. escribed in steps 1 6 of the product protocol Only the 0 ug mL BSA standard standard in Table 1 and 10 ug mL BSA standard standard B in Table 1 are required 2 Transfer 200 uL of each sample into a 0 2 mL thin walled clear PCR tube If using glass mini tubes cat no 22019 100 uL sample can be used for measurement Calibration To move to a previous screen at any time select Return Continue selecting Return to go back to the Main Menu 1 From the AccuLite Main Menu select Calibrate 2 Select AccuOrange from the assay list 3 Insert the blank tube 0 ug mL BSA and close the cover Select Blank 4 The standard value 00010 000 will display Insert the 10 ug mL BSA standard tube and close the cover Press Measure 5 Calibration Finished will appear on the screen 6 Select Return to return back to the Main Menu Sample Measurement 1 From the AccuLite Main Menu select Measure 2 Select AccuOrange from the assay list 3 Insert the first sample tube and close the cover Select Measure The value shown is in ug mL protein 4 Select Save to save the data in the meter Alternatively you can manually the record data without saving then select Return 5 Insert next sample and select Measure 6 After reading all samples select Return repeatedly to navigate back to main menu Retrieving Saved Data 1 From the AccuLite Main Menu select Data 2 Select AccuOrange from the assay list 3 Use the arrow keys to
7. ino acids 10 ug mL DNA 10 ug mL AccuOrange Protein Assay Kit PSF006 2500 400 8 2000 8599 8 NanoOrange S m R 0 9453 o ao 1500 4 0 0 5 1 1 5 5 BSA ug mL S N g S 1000 AccuOrange ue R 0 9981 500 lt AccuOrange CNanoOrange 0 amp T T 0 5 10 15 BSA ug mL Figure 2 BSA titration assayed using AccuOrange Protein Quantitation Kit or NanoOrange Protein Quantitation Kit from Life Technologies according to manufacturer s protocol and read on a microplate reader at the recommended wavelengths for each assay Inset shows the lower end of the curve Error bars represent standard deviation of the mean for triplicate samples Related Products Please visit our website at www biotium com for information on our life science research products including Lumitein protein gel stain environmentally friendly EvaGreen qPCR master mixes fluorescent CF dye antibody conjugates and reactive dyes apoptosis reagents fluorescent probes and kits for cell biology research NanoOrange is a registered trademark of Molecular Probes Inc Materials from Biotium are sold for research use only and are not intended for food drug household or cosmetic use Page 2 of 3 Appendix AccuOrange High Sensitivity Assay Protocol for the AccuLite 470 Fluorometer Sample Preparation 1 Prepare samples and standards as d
8. rd 10 Select Save or is displayed 00010 000 Return if not saving Return t 5 Insert 10 ug mL standard tube select Measure y Calibration Finished Return 6 Select Return once to return to Main Menu Timer Data Value Measure Return 4 1 Insert next sample tube select Measure Repeat for each sample 12 Select Return repeatedly to return to Main Menu 16 Select Return repeatedly to return to Main Menu Figure 4 AccuLite user menu tree showing AccuOrange calibration measurement and data retrieval steps See the AccuLite user manual for complete user menu tree Page 3 of 3
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