Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Concentration of Protein Solutions
Procedures

Below are several Examples of protein concentration using various Methods.

8.5.1.1. Quantitative recovery of Proteins from dilute solutions [396]. To 0.1 ml of protein solution, add 0.4 ml of methanol, mix, and centrifuge for 10 s at 9000 g (applied at this and subsequent steps). Add 0.1 ml of chloroform (0.2 ml for samples with high phospholipid concentrations), mix, and centrifuge for 1 min. Discard the upper layer, add 0.3 ml of methanol to the lower chloroform-containing phase, mix, and centrifuge for 2 min. Separate the supernatant and dry the pellet under a stream of air.

For electrophoretic homogeneity control of the isolated sample, dissolve it in 50–100 µl of SDS (5% solution, w/v) or directly in 50 µl of buffer containing 5% SDS.

This method can be used for both soluble and hydrophobic proteins; the results are unaffected by the presence of detergents, Lipids, salts, or 2-mercaptoethanol in the initial solution. With an initial protein content of 40–120 µg in 0.1 ml, the yields are 92–100%.

8.5.1.2. Electrophoresis using liquid membranes [384, 385]. Electrophoresis with a non-linear pH gradient allows proteins with different charges to be concentrated faster than isoelectric focusing. Two superimposed layers—one consisting of Amino Acids (Histidine or valine) and the other of ampholytes (Servalyte)—form a liquid membrane that acts as a barrier for the protein. Thus, 200 ml of a Ribonuclease solution was concentrated 48.3-fold in 4 h, with a protein recovery of 96.6% [385]. In a system consisting of Pharmalytes (pH 2.5–5.0), histidine (pI 7.64), and ampholytes (pH 9–11), the acidic and basic isoforms of horseradish peroxidase (pI 4.0 and 8.4) were concentrated at two phase boundaries at a voltage of 400 V for 5 h [384].

8.5.1.3. Concentration of microgram quantities of protein [237]. The isolation of microgram quantities of proteins from dilute solutions is based on trichloroacetic acid precipitation with The addition of deoxycholate. The detergent, commonly used in membrane Protein Isolation, is subsequently removed by extraction.

a) Protein precipitation

1. Add 120 µl of sodium deoxycholate (2%, w/v) to the solution to a final protein concentration of 80 µg/ml. Mix and let stand on ice for 30 min. The detergent concentration can vary from 50 to 500 µg/ml without affecting the yield.

2. Add 10 ml of cold trichloroacetic acid (24%, w/v) to a final concentration of 6%. Let stand on ice for 1 h until the protein–detergent complex is completely precipitated.

3. Centrifuge at 2400 g and 4 °C in a swinging-bucket rotor for 45 min.

4. Carefully remove the supernatant by aspiration.

5. Dissolve the pellet in 0.5 ml of 62.5 mM Tris-HCl (pH 6.8) containing 3% SDS, and adjust the solution to 0.5 M with NaHCO₃ (final pH 8.8).

6. Dialyze the solution overnight at room Temperature against 2 l of 6.25 mM Tris-HCl (pH 6.8) containing 0.3% SDS.

7. Lyophilize the solution in screw-capped Glass vials (7 ml) or tubes.

b) Detergent extraction

1. Add 1 ml of extraction mixture (acetone – acetic acid – triethylamine, 90:5:5) to the dried sample.

2. Quickly transfer the solution to a conical centrifuge tube.

3. Let stand on ice for 1 h to precipitate the protein.

4. Centrifuge in a benchtop centrifuge at room temperature.

5. Remove the supernatant by aspiration.

6. Wash the pellet first with 1 ml of the extraction solution and then with 1 ml of acetone.

7. Dry under nitrogen. Yield: 40–80%.