{"text_format":"3","workbook":"xlsx","n_sheets":4,"sheets":["Instructions","Example_Data","Analysis_template","Data_input"],"not_rendered":{"charts":6,"images":2,"macros":1,"defined_names":17},"read":"whole","date_system":"1900"}

{"sheet":"Instructions","index":0,"n_rows":56,"n_cols":1,"rows_kept":56,"hidden":true}
["NECEEM electropherogram batch analysis"]
["Version 0.32 , July 18, 2014"]
["Implementation: Victor Galievsky, Mirzo Kanoatov, Sergey N. Krylov."]
["Based on: Cherney, L.T.; Kanoatov, M.; Krylov, S.N.  Anal. Chem. 2011, 83,  8617-8622"]
["Summary: This program analyzes NECEEM electropherograms and extracts Kd, kon and koff values in a batch regime. It employs the peak deconvolution method developed by Cherney et al (reference 1). This version is developed for analysis of data obtained by a P/ACE MDQ series of Capillary Electrophoresis instruments (Beckman-Coulter), and exported by 32 Karat software, version 7. The program has been written using Visual Basic, and requires Microsoft Excel version 2007 or later. \n\nThe most recent version of this program can be downloaded from: http://www.yorku.ca/skrylov/resources.html\n\nThe general algorithm of the program:  \n- All data files within a user-defined folder are imported into the workbook\n- The baseline is linearised within the user-defined working region of the signal\n- User-defined background regions and a signal-to-noise constant are used to identify non-convoluted edges of the complex and free-ligand peaks\n- The data is smoothed using  Savitzky-Golay method\n- A first derivative of the data is used to approximate maxima of the two peaks. The peak maxima values are further refined by fitting to a polynomial equation. \n- Assuming peak symmetry, convoluted edges of the two peaks are resolved and areas under the three regions of the NECEEM electropherograms are calculated. This step uses the original non-smoothed data\n- The signal areas are used to calculate Kd, kon and koff. "]
[]
["Instructions:"]
["1. Obtain a good set of NECEEM data:\n- Ensure that your Ligand-only peak is symmetric. You should use the symmetry-correcting pressure propagation method described in ref. 2. Also, try to avoid significant mismatches in ionic strengths of sample and run buffers.\n- There should be a sufficient span of baseline both before and after the NECEEM signal. Make sure not to stop the runs prematurely and cut off the end of a peaks.\n- Make sure not to use any internal standards that co-migrate with the NECEEM components. \n- Try to avoid very noisy or irregular data. "]
["2. Group the data for batch analysis:\n-This program works best for batch analysis of replicates and concentration titration experiments. When analyzing multiple results in a batch mode, make sure not to group unrelated data together. \n- In any group of data, the migration times of both the complex and the free ligand peaks should be similar. Thus, do not group data that has been obtained using different capillary lengths, electric field strengths, or using different interacting components. "]
["3. Define the working region and background regions:\n- Open a group of data in 32 Karat software, or an alternative viewing software. Do not adjust the scale or alignment of the traces.  \n- Pick a point in the electropherograms that precedes the NECEEM peaks by a small stretch of the baseline. Pick a value that is common to all of the electropherograms in the group. Record the time value of this point as the beginning of the working region. (Note: 32 Karat displays the time of the point under the cursor in the top left corner of the screen).\n- Pick a point in the electropherograms that follows the NECEEM peaks after a small stretch of the baseline. Pick a value that is common to all of the electropherograms in the group. Record this value as the end of the working region. You now have two points that define the working region.\n- Pick two points within the working region that separate the NECEEM peaks from the baseline. The points should be as close to the NECEEM peaks as possible, but should not enter into the peaks. Record these values as the Edge of background 1 and 2.\n- Make sure to exclude any internal standard peaks or signal abnormalities from the working region.  See example below."]
["Example: The figure below illustrates an example, where 3 replicates of the same experiment are viewed in 32 Karat. "]
{"$rows_empty":32}
["4. Export electropherograms:\n- Use 32 Karats export tool (Method > Advanced > Export tab) to export each of the electropherograms into the ASCII format.\n- If you are not a 32 Karat user,  you can format your result files manually. Just paste the all of the signal values of the electropherogram into the \"Example_Data\" worsheet found in this document. There is no need to define a time column. Define the data acquisition rate (sampling rate) and save the worksheet as a CSV file. \n- Place all of the grouped exported electropherograms into one folder. Do not place any other files into this folder.\n- Use the Windows Explorer address bar to copy the file path to the folder with your data (e.g. C:\\Users\\NECEEM\\Results1\\). This is the Path to data folder."]
["5. Input global parameters\n- Ensure that Macros are enabled in this Workbook.\n- Switch to the \"Data_input\" sheet within this workbook. \n- Fill out the parameters in cells with a light blue highlight.\n \nThe parameters are:\n     - Path to data folder, which you have obtained in step 4.\n     -The initial concentrations of the ligand and the target components, which you have used in your experiment. If you have grouped together data which uses a range of concentrations, enter values of one of the combinations. You will have the chance to update theese values on an individual basis later on. \n     - Ratio of quantum yields of Ligand and Complex. Reference 3 describes a method to obtain this value.\n     - Duration of the pressure propagation step, which you have used in your experiments.\n     - Signal-to-noise ratio coeficient. This parameter should remain at the default value of 5 for clean electropherograms, and increased for noisy electropherograms.\n     - Timepoints that define the working region and background regions, which you have obtained in step 3.\n     - Fractions of peak widths, for complex and ligand, to determine peak maxima. Start with 0.1, and increase up to 0.5 if peak maxima recognition is not working well."]
["6. Analyze data\n- Press the \"Analyze Data\" button.\n- Depending on the number of data files in the group, the calculations may take up to a few minutes. Do not perform any other operations on your computer while the calculations are in progress. An \"Analysis Complete!\" message box will appear when the operation has finished.\n- A summary of your results, including the Kd, kon and koff values, as well as the deconvoluted areas of each NECEEM region, will appear in a \"Results\" table on the right side of the sheet.\n-  If you have grouped together data which uses a range of different concentrations, update the concentration values in the \"Results\" table in the Data_Input worksheet. These values will override the default values in calculation of Kd, kon and koff. "]
["7. Verify accuracy of the results\n- After performing the analysis, additional sheets, Named \"DataSet_n\" will appear in this workbook, where \"n\" refers to the incremental number of each data file that has been processed.\n- Review the charts in each of these sheets, to verify that the program has correctly identified the peak widths and maxima.\n- If any of the values have been misidentified, correct the input parameters on a file-by file basis (only modify cells with a light blue highlight). \n- If your maxima has been missidentified, increase the signal-to-noise ratio coeficient. A value in the range of 3-50 should be sufficient for most cases.\n- Make sure to click the \"Recalculate\" button after the parameter values have been changed. Do not perform any other operations on your computer while the recalculation is in progress. A \"Recalculation Complete!\" message box will appear when the operation has finished."]
["Example: The figure below illustrates results of data processing. Calculated peak maxima are denoted by red and green lines, while peak widths by red and green rectangles."]
[]
["8. Obtain your results\n- Once you are satisfied with the analysis, copy the Results table from the \"Data_input\" sheet into a new Excel file (use the Paste Values operatioin).\n- Press the \"Reset sheet\" button if you wish to erase previous analysis and re-start with a new group of data. "]
[]
[]
["References:"]
["1. Cherney, L.T.; Kanoatov, M.; Krylov, S.N. Method for determination of peak areas in nonequilibrium capillary electrophoresis of equilibrium mixtures. Analytical Chemistry 2011, 83,  8617-8622"]
["2. Kanoatov, M.; Retif, C.; Cherney, L.T.; Krylov, S.N. Peak-shape correction to symmetry for pressure-driven sample injection in capillary electrophoresis. Analytical Chemistry 2012, 84, 149-154"]
["3. Krylova, S.M.; Wegman, D.; Krylov, S.N. Making DNA hybridization assays in capillary electrophoresis quantitative. Analytical Chemistry  2010, 82, 4428-4433"]

{"sheet":"Example_Data","index":1,"n_rows":14714,"n_cols":3,"rows_kept":14714,"hidden":true}
["Data Analysis Template"]
["Please do not change position of elements."]
[]
[]
[]
[]
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["Sampling Rate:",4]
["Total Data Points:",{"$f":"COUNT(A14:A1048576)","v":14701},"< Calculated automatically"]
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{"sheet":"Analysis_template","index":2,"n_rows":64,"n_cols":71,"rows_kept":64,"hidden":true}
["Version:"]
["Maxchannels:",{"$nulls":5},"File name"]
["Sample ID:","Sample "]
["Data File:","C:\\Sample",{"$nulls":65}," Savitzky–Golay smoothing coefficients"]
["Method:","C:\\Sample"]
["User Name:","System",{"$nulls":67},"smooth","dev"]
["Acquisition Date and Time:",{"$nulls":66},{"$f":"(COUNT($BP$8:$BP$46)-1)/2","v":14}]
["Sampling Rate:",4,4,4,"Hz",{"$nulls":62},1,{"$f":"BP8-$BP$7-1","v":-14},{"$f":"3*(3*$BP$7^2+3*$BP$7-1-5*BQ8^2)/(2*$BP$7+3)/(2*$BP$7+1)/(2*$BP$7-1)","v":-0.04338153503893215},{"$f":"3*BQ8/(2*$BP$7+1)/($BP$7+1)/$BP$7","v":-0.006896551724137931}]
["Total Data Points:",14701,14701,14701,"Pts.",{"$nulls":62},2,{"$f":"BP9-$BP$7-1","v":-13},{"$f":"3*(3*$BP$7^2+3*$BP$7-1-5*BQ9^2)/(2*$BP$7+3)/(2*$BP$7+1)/(2*$BP$7-1)","v":-0.026696329254727473},-6.4039408866995075E-3]
["X Axis Title:","Minutes","Minutes","Minutes",{"$nulls":63},3,-12,-0.011247064639723149,-0.005911330049261083]
["Y Axis Title:","RFU","KV","ｵA",{"$nulls":63},4,-11,2.9662588060808304E-3,-0.00541871921182266]
["X Axis Multiplier:",0.016667,0.016667,0.016667,{"$nulls":63},5,-10,0.015943641082684465,-4.9261083743842365E-3]
["Y Axis Multiplier:",{"v":1e-05,"d":"1.00E-05"},{"v":1e-05,"d":"1.00E-05"},{"v":1e-05,"d":"1.00E-05"},{"$nulls":63},6,-9,0.027685082190087747,-0.004433497536945813]
[{"$nulls":67},7,-8,0.03819058212829069,-0.003940886699507389]
[{"$nulls":67},8,-7,0.047460140897293286,-3.4482758620689655E-3]
[{"$nulls":67},9,-6,0.05549375849709554,-2.9556650246305416E-3]
[{"$nulls":67},10,-5,0.062291434927697446,-2.4630541871921183E-3]
[{"$nulls":67},11,-4,0.067853170189099,-1.9704433497536944E-3]
[{"$nulls":67},12,-3,0.0721789642813002,-1.4778325123152708E-3]
[{"$nulls":67},13,-2,0.07526881720430108,-9.8522167487684722E-4]
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{"sheet":"Data_input","index":3,"n_rows":16,"n_cols":14,"rows_kept":16,"merged":[[0,0,0,1],[0,5,0,13]]}
["User Input",null,"Units",null,null,"Results"]
["Path to data folder","C:\\Users\\Mirzok\\Dropbox\\Krylov Lab\\1 Lab tools\\01 Tools\\NECEEM area analysis\\Data Folder\\","File Path",null,"#","File Name","Init. Conc. of Ligand  (M)","Init. Conc. of Target  (M)","Kd (M)","kon (M-1 s-1)","koff (s-1)","Proportion of Free Ligand","Proportion of  Complex","Proportion of Decay"]
[]
["Initial concentration of Ligand",{"v":1E-8,"d":"1.00E-08"},"M"]
["Initial concentration of Target",{"v":1E-8,"d":"1.00E-08"},"M"]
["Quantum yield ratio Ligand/Complex",1]
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[]
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[]
["Peak width fraction for Complex",0.2]
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