Standard Curves For Protein Assays

ByCarrie Trant

I have been working with Barrett to test two different assays with BSA. One is a standard procedure for microtiter plates and the other is a microassay procedure for microtiter plates.

Here is a brief summary of each procedure:

Standard Procedure for Microtiter Plates– linear range is 0.05 mg/ml to 0.5 mg/ml

  1. Prepare a diluted dye reagent that is 1 part Dye Reagent Concentrate to 4 parts DDI water.
  2. Prepare dilutions of a protein standard. I used BSA and did eight dilutions from a stock BSA solution of 2 mg/ml, ending with a concentration of 1/128 mg/ml.
  3. Pipet 10 μl of each standard and a buffer control into separate microtiter plate wells. I used a 96 well plate.
  4. Add 200 μl of diluted dye reagent to each well.
  5. Mix on a microplate mixer for at least 5 minutes.
  6. Measure absorbance at 595 nm. I used the TECAN.

Microassay Procedure for Microtiter Plates– linear range is 8.0 μg/ml to 80 μg/ml

  1. Prepare dilutions of a protein standard. I used BSA and did eight dilutions from a stock BSA solution of 2 mg/ml, ending with a concentration of 1/128 mg/ml.
  2. Pipet 160 μl of each standard and a buffer control into separate microtiter plate wells. I used a 96 well plate.
  3. Add 40 μl of dye reagent concentrate to each well.
  4. Mix on a microplate mixer for at least 5 minutes.
  5. Measure absorbance at 595 nm. I used the TECAN.

I tested each assay five times, in triplicate.

To obtain workable results, I averaged the three rows to obtain an average for each experiment. I zeroed out the buffer by subtracting the buffer’s absorbance reading from each absorbance reading. Then, I averaged the five experiments together to obtain a single curve. I limited the final graphs to the linear range of each assay and did a best fit line, with the positive standard deviations shown.

Results:

Standard Procedure for Microtiter Plates

standard-assay

Microassay Procedure for Microtiter Plates

microassay

The standard procedure gives a much more linear curve, so Barrett and I think we will go with that one for future protein experiments.

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