Protein Purification Using Biotinylated Affinity Molecule Targeting
What is Protein Purification?
Protein purification, the process by which a specific protein is isolated from a biological sample, is a common workflow across numerous life science industries and applications.
Protein purification refers to the process by which a specific protein is isolated from a biological sample. When isolating proteins from tissue samples, this can include the extraction of the sample as well as the breaking down of cell membranes to release the proteins contained within. The target proteins will then need to be separated from the complex biological sample and extracted.
How Does Protein Purification Work?
The way protein purification works depends on the sample type, protein being targeted, and specific isolation method that you choose. Once the sample has been collected, you may need to lyse the cell membranes physically or chemically denature them to release the proteins from the cell system. Once the proteins are freely floating in the sample, you need to separate proteins from the non-proteins. Finally, you will then isolate the desired protein from other surrounding proteins. Each purification step results in cell loss, ideal yield is around 80% each step. Minimizing steps can greatly increase your resulting yield.
Protein Denaturation
Denaturation is an essential step in protein purification because it is used to break down the initial cells or proteins for isolation. Typically, cells will be denatured and the proteins will be captured so as not to damage their structure—because structure dictates the protein’s behavior. However, it is possible to restore some proteins. This means they can be denatured and broken from their initial structure for collection, then reformed into their original structure during testing or application. This can allow for easier capture because the structure of certain proteins will not have to be maintained.
What Happens When a Protein is Denatured?
When a protein is denatured its structure is lost. Many of the bonds and links within a protein molecule are dissolved, causing the structure to be looser. These proteins are typically also insoluble. Depending on the denaturing agent used, it’s possible to keep some structures intact while affecting other structures. For example, egg whites can be denatured in a way that their primary structure holds but their tertiary structure dissolves. The tertiary structure of a protein is the overall three-dimensional positioning of its polypeptide chain. When this structure is lost, the protein will not carry out the same role that it did when fully functional.
Protein Denaturation and Protein Purification
While denaturation can sometimes help you isolate the desired protein, it’s more likely to damage the molecule than regular protein capture methods. Especially when working with fragile or unique proteins, taking the risk of denaturing them can result in a futile experiment. One alternative method of protein capture is to leverage biotinylated affinity molecules that are specific to the protein of interest.
Protein Capture with Biotinylated Affinity Molecules
Biotinylated affinity molecules can be designed to target surface epitopes on specific cells or proteins. This biotinylated affinity molecule, such as a specific antibody, will seek out and bind to the protein of interest, providing a biotin label to each of the proteins you are looking to capture.
Streptavidin Microbubble Protein Purification
Akadeum Life Sciences has developed an innovative cell separation technique that can also be used for protein capture and purification. Akadeum uses hollow, thin-shelled microbubbles to target and isolate labeled cells in a solution. Akadeum’s Streptavidin-coated microbubbles can be used for binding/immobilization and separation of biotin-labeled analytes like proteins from complex samples. Akadeum’s revolutionary, buoyant Streptavidin Microbubbles allow for the use of virtually any biotinylated affinity molecule, providing a flexible and customizable platform capable of meeting highly specific research needs.
Important Protein Purification Metrics
Different purification and denaturation techniques can be used together to provide the best possible process. Some of the key metrics to focus on when purifying proteins include the following:
- Capacity – The number of target proteins that can be loaded if all separation efforts go as planned
- Speed – Very important at the beginning when removing contaminants and trying to prevent cell or protein death
- Recovery – Very important toward the end when the purified product becomes the most important thing; keeping recovery high requires constant monitoring throughout the whole process
- Resolution – The hardest metric to achieve that pertains to selectivity and efficiency of the overall process
How important each parameter is to the process changes depending on the intended result of purification.
Why Are Proteins Denatured and Purified?
Proteins are responsible for virtually every mechanism in every living organism. Proteins are made of amino acid combinations and can be folded into innumerable shapes to perform different tasks.
Akadeum’s Microbubbles
Sample preparation is frequently a critical first step in any number of life science endeavors. Due to the inherent complexity within samples and diversity between applications, sample preparation needs can vary widely. Akadeum’s Streptavidin microbubbles leverage the biotin-streptavidin binding complex combined with the power of buoyancy-based separation in a streamlined workflow that is fast and easy to perform.
Learn more about using Streptavidin for Customized Workflows
Mechanism of Action: Akadeum’s Streptavidin Microbubbles are high-buoyancy microspheres coated in streptavidin. These microbubbles leverage the biotin-streptavidin binding complex and can be used for binding biotinylated target molecules like antibodies, proteins, DNA, and more. They permit the capture and sorting of a diverse range of targets such as cells, bacteria, viruses, or other molecular analytes allowing for highly specific and customized applications.
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