Protein reconstitution or protein dilution is an important part of major laboratory processes. The goal of dilution or reconstitution is to generate a higher quantity of protein without compromising its quality. In addition, protein reconstitution enables increased sample size for downstream applications. For example, diluting a 1ml protein containing 1,000 cells with an equal volume of diluent gives 2ml of protein-diluent mix containing the same 1,000 cells. The resultant mixture can then be subjected to a range of quantitative and qualitative tests based on the specific goal of the research.
If you have read about how to establish a pathological Lab, surely, you must have come across the importance of protein, especially as part of laboratory assays. Below is a guide on how to reconstitute proteins in the laboratory.
The first step in the process of diluting proteins for laboratory assays is protein extraction. The process of protein extraction is dependent on the type of sample you are working on. Plant and animal materials have different methods by which protein can be liberated from them. Some of the common methods used for extracting protein from sample materials include mechanical and detergent-based extraction. Mechanical extraction involves crushing the tissue, organ, or sample to create an increased surface area for the extraction mixture to work on. On the other hand, detergent-based extraction involves using a laboratory detergent to lyse the protein from the sample material. The choice of extraction method will overall affect the quantity and quality of the extracted protein. For example, some extraction methods may offer a higher protein concentration but also contain protein denaturing compounds that may affect the overall result of the extraction.
Once the protein has been successfully lysed from the sample material, the next step is to preserve it to avoid denaturation or damage. Proteins are heat sensitive, thus require a certain temperature below the freezing level to remain intact. Ahead of freezing, the protein can also be stored in different laboratory cocktails and mixtures that contain inhibitor compounds. These compounds prevent denaturation. Some of the common constituents of inhibitor compounds include cysteine, serine, metalloprotease, aminopeptidase, as well as aspartic acid protease.
Different methods of protein extraction offer different levels of protein purity. However, the chance of impurities in your extracted protein exists, and that is why the cleanup stage is important. Protein cleanup can be done using three different methods; Dialysis, Defiltration, and Desalting. The cleanup process prepares the protein to be used for other downstream applications without worrying about skewed results.
The choice of the cleanup process will depend on the downstream application. Therefore, whatever method is chosen has to consider the volume and concentration of protein needed for downstream application, processing time, and functional protein requirements.
After it has been cleaned up, quantifying your target protein can help determine whether you have the right concentration for the task ahead. Quantitation can be done using biochemical assay methods like spectrophotometers, fluorometers, or plate readers. However, note that each of these assay methods has its limitations.
If you have the right concentration but do not have the right quantity of protein for your sample mix, dilution or reconstitution becomes necessary. Depending on your target sample size and quantity, you may mix an equal volume of buffer mix with your protein sample to extend its quantity.