tein Assay: BCA assayst Data analysisUsing the excel format to construct the standard curve for BSA and determine theconcentration of U1-U3123Record the Absorbance values of all standards and unknown samplesAverage the absorbance of all standards and unknown samples on an excelAdjust the avg. of all absorbances by subtracting value of the blank tube (tubes 95)from others (see the reference graph)4Plot concentration (X axis) vs. absorbance (Y' axis) of 9 standard solutions only (15-95)56Select the best straight line on the excel of the graph. Select equationDetermine the concentration of 3 unknown samples by either estimation or using theequation from step 5.
ug/ml at 620 nm O P 2000 1.035 1.098 1.0665 1733 0.889 0.968 0.9285 1466 0.809 0.882 0.8455 1200 0.719 0.722 0.7205 933 0.617 0.616 0.6165 667 0.475 0.513 0.494 400 0.35 0.345 0.3475 133 0.202 0.194 0.198 0 y =0.0005x 0.143 0.15 0.1465 blank tube R2 = 0.9985 0.9 corrected Avg Abs at 620nm corrected ABS is the values of each standard -value of blank tube 0.8 conc (ug/ml) corrected Avg Abs 0.7 2000 0.92 0.6 N 1733 0.782 0.5 1466 0.699 1200 0.574 0.4 A 933 0.47 0.3 6 667 0.3475 0.2 400 0.201 0.1 133 0.0515 9 0 0 500 1000 1500 2000 tubes Abs 1 Abs 2 avg Abs Conc ug/mlcolumns 3 and 4 below. The total volume of each tube is 150ul (BSA and water). The stock concentration is 2000ug/mL. 2 Unknown samples have been prepared by an instructor (done) Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Tubes Final BSA water total Abs conc(ug/ml) vol(ul) vol(ul) vol(ul) 562nm IS 2000 150 0 150 2S 1733 130 20 150 3S 110 40 150 4S 90 60 150 5S 70 80 150 6S 50 100 150 7S 30 120 150 8S 10 140 150 9S 0 150 150 Ul X X 150 U2 X X 150 U3 X X 150 1S-9S represent the standard samples. Each sample has a total of 150ul but you only need 50uL of standard sample to run the experiment. Since we run duplicate you only need 100uL.4. BCA protein assay, 11/22/2022 Set 1 Set 2 samples Label samples Working Samples Working Samples Absorbance Samples reagent Volume reagent Volume (ul) at 562 mmi Tubes Volume (ul) Volume Cul (ul) 400 50 400 50 1rx 400 50 400 50 2rx 400 50 400 50 31x 400 50 400 50 4rx 400 50 400 50 5rx 400 50 400 50 61X 400 50 400 50 71x 400 50 400 50 Six 400 50 400 50 9rx 400 50 400 50 Ulrx 400 50 400 50 U2rx 400 50 400 50 U3rx Incubate 37C for 30 minutes Transfer 250ul of all standard and unknown samples (tubes rx) to 96 wells plates in an order (left to right) row A (1rx, 2rx. ......U3rx) Repeat step 4 for row B Absorbance readings for rows A and B are duplicated. Measure the absorbance at or near 562 on a plate reader400 50 400 50 rx 400 50 400 50 2rx 400 50 400 50 31x 400 50 400 50 4rx 400 50 400 50 5rx 400 50 400 50 61X 400 50 400 50 71X 400 50 400 50 8rx 400 50 400 50 91X 400 50 400 50 Ulix 400 50 400 50 U2rx 400 50 400 50 U3rx Incubate 37C for 30 minutes Transfer 250ul of all standard and unknown samples (tubes rx) to 96 wells pla in an order (left to right) row A (1rx, 2rx.......U3rx) Repeat step 4 for row B Absorbance readings for rows A and B are duplicated Measure the absorbance at or near 562 on a plate reader Post Data analysis Using excel format to construct the standard curve for BSA and determine the concentration of U1-U3 Record the Absorbance value for all standards and unknown samples WNE Average the absorbance of all standards and unknown samples on an excel Plot Absorbance value (Y coordinate) vs. concentration (X coordinate ) of standards (tubes 1-9) 4 Select the best straight line on the excel\f1. BCA protein assay. 11/22/2022 Name: Lab: Pierce BCA Protein Assay Kit source: Thermo-Scientific Catalog: 23225 Purpose: To determine protein concentration of BSA using Pierce BCA reagent To prepare standard solutions of BSA To determine the concentration of unknown (BSA) To graph Absorbance vs concentration on excel or manually A. Preparation of standards (BSA) stock = 2000ug/ml 1 Prepare 9 standard samples according to the table below in 1.5ml centrifuge tube of columns 3 and 4 below. The total volume of each tube is 150uL (BSA and water). The stock concentration is 2000ug/ml 2 Unknown samples have been prepared by an instructor (done) Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Tubes Final BSA water total Abs conc(ug/ml) vol(ul) vol(ul) vol(ul) 562mm IS 2000 150 0 150 2S 1733 130 20 150 3S 110 40 150 4S 90 60 150 5S 70 80 150 6S 50 100 150 7S 30 120 150 8S 10 140 150 95 0 150 150 U1 X X 150 U2 X X 150 U3 X X 150 1S-9S represent the standard samples. Each sample has a total of 150ul but you only need 50ul of standard sample to run the experiment. Since we run duplicate you only need 100uL
