How can I resuspend protein pellet after TCA extraction and which methods can I use for protein quantification?

Malcolm Nobre

Hello Maria Letizia Tramarin

You may resuspend the pellet in 1-2ml of PBS and 50µl of 8% phenylmethylsulfonyl fluoride (PMSF) (protease inhibitor).

You may use the Bicinchoninic Acid (BCA) assay, a commonly used assay, as it is known for its robustness.

You may want to refer to the article attached below.

Article Bioprocess monitoring: minimizing sample matrix effects for ...

Best.

Bandla Ramesh

Resuspending a protein pellet after a trichloroacetic acid (TCA) extraction and quantifying the protein concentration are important steps in many biochemical and proteomic experiments. Here's a general guide on how to resuspend the protein pellet and methods for protein quantification:

Resuspending the Protein Pellet:

Wash the Pellet: After the TCA precipitation, you'll usually have a protein pellet at the bottom of your tube. Carefully decant the supernatant to remove any residual TCA.

Add a Resuspension Buffer: To resuspend the protein pellet, you can use a suitable buffer. The choice of buffer may depend on your downstream applications. Common choices include: SDS-PAGE Sample Buffer: If you're planning to perform SDS-PAGE, you can resuspend the pellet in a buffer containing SDS, such as Laemmli sample buffer. This will denature the proteins. Urea Buffer: Urea is commonly used for denaturing and solubilizing proteins. A typical urea buffer contains urea, Tris-HCl, and dithiothreitol (DTT) or β-mercaptoethanol to reduce disulfide bonds. Phosphate-Buffered Saline (PBS): If you want to maintain the native structure of your proteins, you can resuspend the pellet in PBS or a similar isotonic buffer.

Vortex and Sonicate: After adding the appropriate buffer, vortex the tube to mix the contents. You may also need to sonicate the sample for a brief period to ensure complete solubilization. Be cautious not to over-sonicate, as excessive heat can damage proteins.

Incubate: You can incubate the sample at room temperature or on ice for a period of time to facilitate protein solubilization. The duration of incubation may vary based on the type of buffer and the nature of your proteins.

Centrifugation (Optional): If there are any insoluble particles or debris left in the sample after resuspension, you can centrifuge it at a low speed to pellet these impurities and then transfer the clear supernatant to a new tube.

Protein Quantification:

To determine the protein concentration of your sample, there are several methods available. Here are a few common ones:

Bradford Assay: The Bradford assay is a colorimetric method that utilizes Coomassie Brilliant Blue G-250 to bind to proteins, causing a color change. The intensity of the color is proportional to the protein concentration.

Bicinchoninic Acid (BCA) Assay: The BCA assay is another colorimetric method that relies on the reduction of Cu²⁺ ions by proteins in an alkaline medium. The resulting purple color can be quantified spectrophotometrically.

Lowry Assay: The Lowry assay is a more sensitive protein quantification method that involves a reaction between proteins and a mixture of Folin-Ciocalteu reagent and phosphomolybdotungstic acid. It results in a blue color, which can be measured spectrophotometrically.

UV Absorbance at 280 nm: Proteins absorb UV light at 280 nm due to the presence of aromatic amino acids. You can estimate protein concentration by measuring the absorbance at 280 nm, although this method is less specific than the colorimetric assays mentioned above.

Fluorescence-Based Assays: Some fluorescent dyes, such as SYPRO Orange or SYBR Green, can be used for protein quantification. These dyes bind to proteins and emit fluorescence, which can be quantified using a fluorescence spectrophotometer.

Choose the quantification method that best suits your specific needs and the equipment you have available in your laboratory. Ensure your measurements are in the linear range of the chosen assay and perform appropriate dilutions if necessary.

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