Molecular Biology. A Practical Guide - Velikov V.A. 2013

Determination of Protein Concentration
Protein Concentration by TCA Precipitation

The trichloroacetic acid protein precipitation method is useful when the protein concentration in a colloidal solution is too low for analysis, or the sample volume is too large to load the required amount of protein into a gel well for Electrophoresis. The pellet is redissolved in a smaller volume of buffer, thereby concentrating the protein solution.

Materials and Equipment

TCA, sodium deoxycholate, BSA protein solution, test protein solution, centrifuge.

Solutions

- Trichloroacetic acid (TCA).

- 0.15% sodium deoxycholate. Store at room Temperature.

Procedure

1. If the minimum protein concentration is around 5 µg/mL, add 1/10 of the volume of 100% TCA to the protein solution.

2. If the minimum protein concentration is less than 1 µg/mL, add 1/10 of the volume of 0.15% sodium deoxycholate (coprecipitant) to the protein solution, incubate for 10 min at room temperature, and add 1/20 of the initial volume of 100% TCA.

3. Incubate for 30 min on ice or 15 min at -20°C.

4. Centrifuge for 5-10 min at maximum centrifuge speed.

5. Remove the supernatant with a pipette. The protein is in the pellet.

6. The pellet can then be dissolved in 50-100 µL of 0.1 N NaOH and washed with 1 mL of a 1:1 ethanol-ether mixture. To completely remove residual TCA, washing must be performed thoroughly and repeated several times.

7. Centrifuge at maximum microcentrifuge speed. Discard the supernatant. Dissolve the protein pellet in PBS buffer.