Precipitation experiment with trichloroacetic acid

Summary

TCA precipitation experiments are mainly used for (1) precipitation purification of proteins and (2) extraction of specimen compounds.

Operation method

Precipitation experiment with trichloroacetic acid

Principle

1. TCA is an acidic compound and an anionic precipitant, when the pH value is lower than the isoelectric point of proteins, the acid root forms insoluble salts with proteins with positive point charge and precipitates; 2. As a protein denaturing agent, protein conformation is changed and more hydrophobic groups are exposed, which leads to aggregation and precipitation; 3. As the molecular weight of proteins increases, the more structural complexity and denseness of proteins are greater, and TCA may penetrate into the molecules and make it more difficult to be completely removed. As the molecular weight of the protein increases, its structure becomes more complex and dense, TCA may penetrate into the molecule and make it more difficult to be completely removed, and when the sample is heated before electrophoresis, acid hydrolysis of the protein structure may occur and fragmentation may occur, and this effect becomes more and more obvious with the prolongation of time.

Materials and Instruments

Protein Sample
Acetone Molecular Weight Standard TCA+DOC with and without 2-mercaptoethanol Sample Buffer
Polypropylene Microcentrifuge Tubes

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MATERIALS AND EQUIPMENT

Acetone (Aldrich Chemical Co. Inc.)
NOTE: Store at room temperature. It should not be placed in the refrigerator.

Polypropylene Microcentrifuge Tubes (1.5-ml)
NOTE: 1. Wash the tubes before use. 2. When analyzing very small quantities of proteins (<100 ng) it may be necessary to silylate the tubes in the following way: the tubes are briefly (5 min) immersed in a 5% (v/v) dichlorodimethylsilane (Aldrich Chemkai Co., Inc.) vinyl chloride solution. and then washed with ethanol (100%) and glass distilled water. Centrifuge tubes were dried at room temperature for at least 1 day, and some proteins bound more tightly to polypropylene after silanization. Unfortunately this property is relatively empirical and it is not possible to know in advance whether the proteins to be measured will remain on the washed polypropylene or the walls of the silanized centrifuge tubes.

Molecular Weight Standards (optional)

Reagents and Buffers

TCA+DOC

Sample Buffer (1X) with and without 2-mercaptoethanol

(for recipe, see "Preparation of Reagents and Buffers," P.262)

Operating Procedures

1) Add 1/4 volume of TCA+DOC to the Protein Fractions in a 1.5-ml polypropylene microcentrifuge tube to a final concentration of 20% (w/v) TCA. Shake to mix.

2) Dissolve on ice for 20-30 min.

3) Microcentrifuge at room temperature for 15 min. Precipitate, if visible, is a viscous, yellowish-brownish gel. Aspirate the supernatant with a fine pasteurized pipette. Try to remove as much of the supernatant as possible. If a 100ul sample is taken for precipitation, the precipitate will be visible.

4) Add 3 times the volume (original sample volume) of acetone (room temperature). The sample was allowed to stand at room temperature for about 10 min to allow the TCA+DOC to dissolve in the acetone.

5) Centrifuge the sample at room temperature for 15 min. At that time, the size and physical properties of the precipitate resembled a bit of dust. About 10ug or a little more of protein can be seen. Sometimes, a white precipitate of salts (e.g., KCl, etc.) is obtained. The supernatant is removed with a very fine pasteurized pipette. The precipitate is dried on ice for 10 min (with the lid of the 1.5-ml centrifuge tube open). The dried precipitate can be stored at -20°C for extended periods of time (>1 month).

6) Dissolve the precipitate with Sample Buffer (lx) containing 2-mercaptoethanol (6ul for a standard small plate gel setup). If a small amount of TCA residue remains, the bromophenol blue will turn yellow. This does not matter because there is enough buffering capacity in electrophoresis to neutralize the trace TCA. if there is residual KCl in the precipitate, a flocculent white precipitate (potassium dodecyl sulfate) will form when sample buffer is added to the precipitated protein. If potassium dodecyl sulfate precipitates, it is difficult to add the sample to the SDS gel, but once the sample is added to the gel, electrophoresis proceeds well.

7) If a molecular weight standard reference is required, a protein molecular weight (MW) standard can be prepared in lxSDS Sample Buffer, which does not contain 2-mercaptoethanol, as a reservoir of 0.5 mg of total protein per milliliter. Divide into small aliquots (50ul) and store at -20°C. For silver staining, take 2ul of the standard. For silver staining, take 2ul of Molecular Weight Standard + 4ul of Sample Buffer with 2-mercaptoethanol to a final volume of 6ul.

Caveat

1. In the protein precipitation experiment, shake well so that the TCA can fully bind to the protein in the solution.

2. Incubate at room temperature for 10 min.

Common Problems

TCA is the most effective protein precipitant, but the reagent often contains impurities, resulting in high blank values in the method, followed by the fact that if ether is used as the extraction solvent after the proteins are extracted, TCA will be dissolved in the ether to interfere with the determination.

This experiment describes the process of TCA precipitation method. This experiment is from the Experimental Guide for Protein Purification and Identification, by Zhu Houzhu.

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