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{"sheet":"ACID PHOSPHATASE ASSAY","index":0,"n_rows":55,"n_cols":23,"rows_kept":55,"cut":false,"hidden":false,"merged":[[21,9,21,14],[21,15,21,18],[21,3,21,6],[21,21,21,22],[1,17,1,19],[21,8,22,8],[21,7,22,7],[20,7,20,14],[22,9,22,10],[22,11,22,12],[22,13,22,14]],"flags":["formulas_cached_only"]}
[]
[null,null,"General Enzyme Calculation Template",{"$nulls":13},"Date","2009-05-10T15:01:03.720"]
[{"$nulls":4},"-determine protein concentration of samples using ProteinMicroassay or ProteinMicrotiterPlateassay sheet"]
[{"$nulls":4},"-determine activity of samples using standard curve calibration assay below to obtain Total Units per mL"]
[{"$nulls":4},"-divide Total Units per mL by Protein (mg/ml) to obtain specific acitivity"]
[]
[{"$nulls":4},"A.      Enzyme assay "]
[{"$nulls":4},"1.  Prepare reaction buffer containing : 250 mM glycine/HCl buffer with 25 mM p-nitrophenylphosphate (PNPP)"]
[{"$nulls":4},"2. Prepare 3 tubes, labelled with (1. Control   2. WT  3. rpoS )"]
[{"$nulls":4},"3. Add 0.5 ml reaction buffer into each tube"]
[{"$nulls":4},"4.  Incubate all three tubes in water bath at 37oC for 2 min to equilibrate"]
[{"$nulls":4},"5. Start the reaction by adding 20 µl cell extract (if the protein concentration is higher than 1 mg/ml) of WT and rpoS to tube 2 and 3, respectively. Add  20 µl of Pi buffer to control tube 1"]
[{"$nulls":4},"6. Incubate at 37oC for 15 min and stop the reaction by adding 1 ml of 1N NaOH  (TOTAL Volume = 1.52 mL)"]
[{"$nulls":4},"7. Transfer 1 ml reaction mixture to cuvette to measure absorbance at  410 nm "]
[]
[{"$nulls":4},"Assay Notes  :      (1) make sure that you perform linearity checks"]
[{"$nulls":6},"(2) use only determine unknown values with in the standard curve (do not extrapolate..dilute and re-assay instead)"]
[{"$nulls":6},"(3) biological replicates usually yields standard errors of less than 10% (5% is quite good). If yours is higher check your technique.."]
[]
[]
[{"$nulls":7},"ACID PHOSPHATASE ASSAY DATA"]
[null,null,null,"Protein concentration (mg/mL)",null,null,null,"Volume of Sample (ul)","Time of Incubation(min)","OD410 READINGS",{"$nulls":5}," Total Activity (umol/min/mL)",{"$nulls":5},"Error Flags"]
[null,"Sample ","Sample ID","Rep1","Rep2","Rep3","Average",null,null,"Rep1",null,"Rep2",null,"Rep3",null,"Rep 1","Rep 2","Rep 3","Average OD410","SPECIFIC ACTIVITY","Standard Error",0.1,0.2]
[{"$nulls":9},"Sample1","Sample2","Sample1","Sample2","Sample1","Sample2"]
[null,1,"n",null,null,null,{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,2,{"$nulls":4},{"$error":"#DIV/0!"}," ",{"$nulls":7},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,3,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,4,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,5,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,6,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,7,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,8,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,9,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,10,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,11,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,12,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,13,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,14,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,15,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,16,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,17,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,18,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,19,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[null,20,{"$nulls":4},{"$error":"#DIV/0!"},{"$nulls":8},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"},{"$error":"#DIV/0!"}]
[]
[null,"CALCULATION NOTES",{"$nulls":16},"=-user inputed data"]
[null,"Protein Concentration (from Protein Assay Table)",{"$nulls":16},"= spreadsheet calculated data"]
[null,"Rep - Biological Replicate of Experimental unit",{"$nulls":16},"= negative number (this occur if blank values are  higher than measured values)"]
[null,"Sample - Technical Replicate",{"$nulls":16}," error flag - excess variation "]
[null,"TOTAL ACTIVITY = (AverageOD410/1770 M-1) x (10E9 nM/M) x (1.0 l/1000mL) x 1.52mL (total assay volume) x (1/sample volume(mL)) x ((1/incubation time) (min))",{"$nulls":16},"error flag - very high varation that will likely affect ability to "]
[null,"SPECIFIC ACTIVITY = TOTAL ACTIVITY/PROTEIN CONC. (e.g. P25/D25)",{"$nulls":17},"employ statistical tests of significance"]
[null,"STANDARD ERROR = STANDARD DEVIATION of Biological Replicate Specific Activities (e.g. Standard Deviation (P25/D25, Q25/E25, R25/F25) / SQRT(3))"]
[null,"ERROR FLAGS - these will indicate if variation is high. If high, check to make sure numbers are typed correctly--it is easy to mistype a decimal)"]
[null,null,"           -this is not operational"]
[null,"MOLAR EXTINCTION COEFFICIENT ( e ) OF pPNP = 17,700 M-1 cm-1 "]

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[]
[null,"Bradford Protein Assay (MICROASSAY)",{"$nulls":6},"Date","2009-05-10T15:01:03.720"]
[null,null,"-add water to standard tubes in triplicate",{"$nulls":5},"User"]
[null,null,"-add protein standard volume to each tube, use a fesh tip for each tube (total volume)"]
[null,null,"-add Protein dye reagent (200ul) to each tube: allow 15s between additions"]
[null,null,"-mix thoroughly with a vortex mixer"]
[null,null,"-incubate at least 5 min (preferably 30min) and read at 595 in the same order as protein reagent addition"]
[null,null,"Notes: (1) the \"0\" protein control as it is used for both the standard curve and for background "]
[null,null,"                subtraction (make sure calculated and measured values are in good agreement)"]
[null,null,"           (2) make sure that all unknown values fall in the linear portion of the curve--if not, dilute and reassay"]
[null,null,"           (3) if possible, use Corrected A595 (Average-blank) values in the range 0.01 to 0.400"]
[null,null,"           (4) if the calculated Intercept differs from the Corrected for Blank (below) by "]
[null,null,"                more than .005, check standard curve"]
[null,null,"           (5) Use 1.0 mg/mL BSA Standard"]
[null,"Standard Curve",{"$nulls":15},"Regression"]
[]
[null,"BSA Protein (ug)","Volume of ","Volume of","Total Vol.","Abs At 595 nm (triplicate)",null,null,"Ave.        (595 nm)","Corrected for Blank  (595 nm)",{"$nulls":7},"Slope",{"$error":"#DIV/0!"}]
[null,null,"Standard (ul)","Water (ul)",{"$nulls":13},"Intercept",{"$error":"#DIV/0!"}]
[null,0,null,null,800,null,null,null,"",""]
[null,2,null,null,800,null,null,null,"",""]
[null,4,null,null,800,null,null,null,"",""]
[null,6,null,null,800,null,null,null,"",""]
[null,8,null,null,800,null,null,null,"",""]
[null,10,null,null,800,null,null,null,"",""]
[]
[]
[]
[null,"Unknown Samples"]
[]
[null,"Sample Number","SampleID","Volume of Sample (ul)","Volume of Water (ul)","Readings",null,"Average","Average-blank","Protein (mg/mL)","Error Flags"]
[{"$nulls":5},"Rep #1","Rep#2",null,null,null,0.025,0.05," "]
[null,1,{"$nulls":5},"",""," ",null,null," ",null,"- User input"]
[null,2,{"$nulls":5},"","","",null,null," ",null,"- Spreadsheet calculated values"]
[null,3,{"$nulls":5},"","","",{"$nulls":4},"- Protein value wrong--check standard curve"]
[null,4,{"$nulls":5},"","",""]
[null,5,{"$nulls":5},"","",""]
[null,6,{"$nulls":5},"","","",{"$nulls":6}," "]
[null,7,{"$nulls":5},"","",""]
[null,8,{"$nulls":5},"","",""]
[null,9,{"$nulls":5},"","",""]
[null,10,{"$nulls":5},"","",""]
[null,11,{"$nulls":5},"","",""]
[null,12,{"$nulls":5},"","",""]
[null,13,{"$nulls":5},"","",""]
[null,14,{"$nulls":5},"","",""]
[null,15,{"$nulls":5},"","",""]
[null,16,{"$nulls":5},"","",""]
[null,17,{"$nulls":5},"","",""]
[null,18,{"$nulls":5},"","",""]
[null,19,{"$nulls":5},"","",""]
[null,20,{"$nulls":5},"","",""]
[]
[null,"NOTES"]
[null,"Average- Blank - The average corrected for background which is defined as the Standard sample at 0 protein."]
[null,"Error Flags not functional yet"]