Accurate Protein Assay Methods

A) Measure proteins in preparation for enzyme assays

B) Standardized extracts to the same protein concentration (i.e. for equal protein loading for Western blot or ELISA)

C) Measure protein serum or tissue extracts (i.e. high concentration protein solutions,10-100 mg/ml)

 

A) Measure proteins in preparation for enzyme assays

  1. Label two 1.5 ml Eppendorf tubes for each sample of protein plus one pair for the null protein control.
  2. Pipette 10 μl of protein solution into each tube and the control buffer into the null protein control tube..
  3. Pipette 1 ml of ADV01 or ADV02 into the tube containing the protein solution and mix by inverting.
  4. Incubate at room temperature for one minute.
  5. Blank the spectrophotometer on the null protein control sample at 600 nm.
  6. Read each sample and record the values.
  7. Type the results into Excel and compute the average OD600nm of each pair (i.e. "=average(cell1:cell2)").
  8. Multiply the average OD600nm by 30 (ADV01) or 10 (ADV01) to determine the concentration in mg / ml.
  9. Using the equation x = [(A-B) / B] x V = Volume of buffer to add, to make the protein solution into the correct concentration.

Where

A = protein concentration of the sample, e.g. 780 µg/ml

B = required protein concentration, e.g. 100 µg/ml.

V = volume of original sample, e.g. 500 µl.

Volume of buffer to add:  [(780-100) / 100] x 500 = 3400.µl.

 

B) Standardize extracts to the same concentration (useful for quantitative western blotting and equivalent GLISA or ELISA loadings)

  1. Label two 1.5 ml Eppendorf tubes for each sample of protein plus one pair for the null protein control.
  2. Pipette 10 μl of protein solution into each tube and the control buffer into the null protein control tube..
  3. Pipette 1 ml of ADV01 or ADV02 into the tube containing the protein solution and mix by inverting.
  4. Incubate at room temperature for one minute.
  5. Blank the spectrophotometer on the null protein control sample at 600 nm.
  6. Read each sample and record the values.
  7. Type the results into Excel and compute the average OD600nm of each pair (i.e. "=average(cell1:cell2)").
  8. Multiply the average OD600nm by 30 (ADV01) or 10 (ADV01) to determine the concentration in mg / ml.
  9. Using the equation x = [(A-B) / B] x V = Volume of buffer to add, to make the protein solution into the correct concentration.

Where

A = the protein concentration of the samples which are higher concentration than the lowest, e.g. 350 to 780 µg/ml

B = the lowest protein concentration extract, e.g. 250 µg/ml.

V = volume of original sample, e.g. 500 µl.

Volume of buffer to add to extract sample:  [(780-250) / 250] x 500 = 1060.µl.

 

C) High concentration protein solutions using ADV02 (10-100 mg/ml):

1. Label two 15 ml polypropylene screw cap tubes for each sample.

2. Pipette 10 μl of each protein solution onto the wall of their respective labeled 15 ml tubes.

2. Pipette 5 ml of ADV02 onto the same location as the protein solution, cap and tip up and down five times.

3. Incubate at room temperature for one minute.

4. Blank the spectrophotometer on 1 x ADV02 and read absorbance of your sample at 600 nm.

5. Calculate protein concentration based on 1.00 OD600 nm  = 50 mg/ml protein solution.