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75 ul (microassay) / 10μL (microplate)
BCA Working Solution: Use the following formula to determine the total volume or Working Solution required:(standards + samples) x (replicates) x (volume of Working Solution per sample) = Total volume Working Solution Required 50:1 (v/v) Reagent A/ Reagent B. Prepare fresh daily
MICROASSAY:1.Prepare the calibrate in 1.5 mL tubes following the Table 2 (see kit booklet). For the diluent, use the same buffer as in the samples. 2.Pipette 75 μL of each standard or unknown sample solution into separate 1.5 mL tubes.3. To each tube, add 1425 μL of freshly prepared BCA Working Solution. Incubate the tubes 15 min at 60 °C. 4.Cool all samples to room temperature.5. Mix the samples, zero the spectrophotometer with the blank and measure the absorbance at 562 nm.MICROPLATE: 1.Prepare the calibrate in 1.5 mL tubes following the Table 3. For the diluent use the same buffer as in the samples. 2.Pipette 10 μL of each standard and unknown sample replicate into a microplate well. Refer to the Table 3 as a guide for diluting the protein standard (See kit booklet). For the diluent, use the same buffer as in the samples. 3.Add 200 μL of freshly prepared BCA Working Solution and immediately mix the microplate on plate mixer for 30 seconds. 4.Cover and incubate the microplate 15 min at 60 °C. 5. Cool plate to room temperature. 6.Mix the samples, zero the spectrophotometer with the blank and measure the absorbance at 562 nm.
1.If the spectrophotometer or microplate reader was not zeroed with the blank, then average the blank values and substract the average blank value from the standard and unknown sample values. 2.Create a standard curve by plotting O.D. 562nm (y-axis) vs standard, μg (x-axis). Determine the unknown sample concentration using the standard curve. 3.The level of detection of the assay is lower for the microplate assay when compared with the microassay due to a shorter light path used in the microplate reader. 4.Standard curve example for microplate assay procedure is shown in Figure 3. (See Images) 5.Measure the absorbance of these standards, blanks and unknown samples at 562nm.