Experimental Procedures for Protein Estimation

1. Bradford Protein Assay

A. Preparation of Bradford Reagent

  1. Weigh 100 mg of Coomassie Brilliant Blue G-250 dye.
  2. Transfer the dye into a clean flask.
  3. Add 50 mL of ethanol to the flask.
  4. Add phosphoric acid to the same flask.
  5. Shake the flask thoroughly to dissolve the dye.
  6. Transfer the solution into a beaker.
  7. Add distilled water to make the final volume 1 L.
  8. Mix well. The Bradford reagent is now ready for use.

B. Preparation of Standard Samples

  1. Select the standard samples as per the given table.
  2. Set the micropipette to 50 µL and pipette the BSA sample.
  3. Set the micropipette to 450 µL and add distilled water.
  4. Set the micropipette to 2500 µL (2.5 mL) and add Bradford reagent.
  5. Click the Run option to prepare remaining standard samples automatically.

C. Measurement of Absorbance at 595 nm

  1. Transfer the blank sample into a cuvette using a micropipette.
  2. Insert the cuvette into the spectrophotometer.
  3. Set the instrument to Blank mode.
  4. Press A595 to record blank absorbance.
  5. Transfer the test sample into a cuvette.
  6. Insert the cuvette into the spectrophotometer.
  7. Set the instrument to Test mode.
  8. Press A595 to record test absorbance.

D. Measurement of Standard Samples

  1. Repeat the absorbance procedure for all standard samples at 595 nm using the Run button.

2. Lowry Protein Assay

A. Preparation of Standard Samples

  1. Set micropipette to 0.2 mL and add BSA solution.
  2. Click Run to prepare remaining BSA standards.
  3. Set micropipette to 0.8 mL and add distilled water.
  4. Click Run to complete water addition.
  5. Set micropipette to 4 mL and add AC reagent.
  6. Click Run to add AC reagent to all samples.
  7. Incubate for 10 minutes at room temperature.
  8. Set micropipette to 0.4 mL and add FC reagent.
  9. Click Run to add FC reagent.
  10. Incubate for 15 minutes at room temperature.

B. Measurement of Absorbance at 660 nm

Blank Sample

  1. Set micropipette to 1 mL.
  2. Transfer blank sample into a cuvette.
  3. Insert the cuvette into spectrophotometer.
  4. Set to Blank mode.
  5. Press A660 to record absorbance.

Test Sample

  1. Set micropipette to 1 mL.
  2. Transfer test sample into a cuvette.
  3. Insert the cuvette into spectrophotometer.
  4. Set to Test mode.
  5. Press A660 to record absorbance.

C. Measurement of Standard Samples

  1. Set micropipette to 1 mL.
  2. Click Run to measure all standard sample absorbance at 660 nm.
  3. Calculate the concentration of the test sample.

3. BCA Protein Assay

A. Preparation of Standard Samples

  1. Select the standard samples as per table.
  2. Set micropipette to 0.2 mL and add BSA sample.
  3. Set micropipette to 0.8 mL and add distilled water.
  4. Set micropipette to 2 mL and add BCA reagent.
  5. Click Run to prepare remaining standard samples.

B. Measurement of Absorbance at 562 nm

Blank Sample

  1. Transfer blank sample into a cuvette.
  2. Insert the cuvette into spectrophotometer.
  3. Set to Blank mode.
  4. Press A562 to record absorbance.

Test Sample

  1. Transfer test sample into a cuvette.
  2. Insert the cuvette into spectrophotometer.
  3. Set to Test mode.
  4. Press A562 to record absorbance.

C. Measurement of Standard Samples

  1. Repeat absorbance measurement for all standard samples at 562 nm using the Run button.
  2. Calculate the concentration of the unknown protein sample.