The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration. It is important to measure protein concentration after performing a protein extraction or purification, and prior to any type of labeling procedure. The protein concentration after extraction or purification may provide information about a biochemical pathway or a disease state. All commercially available proteins are accompanied by a product information sheet that has the results of a protein quantification method. This is particular important in antibody validation. It is important to know the protein concentration prior to any labeling step so you can ensure that the stoichiometric ratio between label and protein is optimal for clean and efficient labeling. It is equally important to know how much protein you are working with when designing biosensors so that you can define limits of detection and instrument sensitivity.
What is process validation and why do we do process validation?
Good manufacturing practice is an essential part of the production of human drugs, veterinary drugs, biological and biotechnology products, and pharmaceutical ingredients. These commercial processes are subject to regulatory oversight and must ensure that every aspect of the production process is carefully scrutinized. The purpose of process validation is to collect data and scientifically analyze the production process from conception to large scale production. An updated process validation protocol is essential to ensuring product quality and consistency. Many laws have been established to mandate process validation in order to protect consumers, especially in the case of pharmaceutical products.
Process validation in the pharmaceutical industry takes the same form as process validation in all other industries, but the stakes are higher because the product is made for human consumption. Moreover, pharmaceuticals are made to alter the natural biochemical pathways in the human body, so these chemicals must be formulated correctly and consistently every time. Additionally, the products must be stored properly and shipped under climate-controlled conditions in order to ensure efficacy once reaching the consumer.
The ELISA (Enzyme Linked ImmunoSorbent Assay) is the gold star immunoassay, which means that it is the standard procedure that all new assay technology is compared to during research and development. The ELISA is also fundamental to most clinical tests for diagnosis of disease because it is currently the most characterized and standardized method. The ELISA is an immunoassay, the principle of which relies on the specific recognition between an antibody and antigen. This specificity comes from the unique three dimensional structure of the antibody paratope and the antigen epitope. These two regions fit like a lock and key via non-covalent, charge-based, and/or hydrophobic interactions. The clinical purpose of the ELISA is to detect either antibody or antigen from a biological fluid such as blood (serum), urine, or saliva. When the ELISA is used to antibody, the assay is being used to assess whether or not the patient has been exposed to a certain antigen at some point. It is difficult to assess current infection with this method because the body retains antibodies forever after the first introduction. However, elevated amounts of antibody can be indicative of active immune response to the pathogen. One major benefit of the ELISA is that it is quantitative, meaning that an actually number of protein can be evaluated. When the ELISA is used to detect antigen it provides a better understanding of current infection since the antigen would be cleared if it was no longer active in the body.
Biotinylation kit means attaching a biotin tag to a molecule. Biotin is a natural molecule that is also known as vitamin B7. It is an important component in a healthy diet, but it is also very useful in the laboratory as a method for protein conjugation. In the laboratory, he purpose of biotinylation is to create a controlled site for biotin-streptavidin affinity binding.
What is biotin labeling and how does streptavidin bind biotin?
The biotin fits exquisitely into a biotin-binding pocket in each of the four binding sites per streptavidin molecule, and it is held in place with hydrogen bonds. Additionally, once the biotin is bound, a conformational change in the streptavidin allows a small “cap” to close over the biotin in the binding pocket. As a result, biotin and streptavidin have an extraordinary affinity for each other (Kd=10^-15). With such a low dissociation constant, once the biotin and streptavidin are bound it is unlikely that they will dissociate. This affinity is resistant to changes in temperature, pH, and salt concentration and is extremely specific. It is often thought of as a nearly covalent bond. These properties make biotinylation a useful tool for engineers who are developing new purification and detection methods. A commercially available biotinylation kit makes the process even easier.
Antibodies are a key component to many biotechnical applications. They are most often used for immunoassays such as ELISA, cell and tissue staining, protein quantification such as western blot, and cutting edge sensor development. Verified antibodies are easily purchased from commercial vendors. These antibodies can be monoclonal or polyclonal, and can come as a lyophilized powder or as a premixed solution. All of these details must be considered when choosing which antibody to purchase because they all have an effect on the antibody concentration and dilution process.
The difference between IP and coIP
Co-immunoprecipitation (coIP) is a protein extraction technique that specifically targets protein-protein interactions. It is slightly different from immunoprecipitation. Immunoprecipitation utilizes antibodies immobilized on a mobile support to capture target proteins. Co IP protocol takes this concept one step further by using antibodies to target not only the direct antigen that binds to the antibody, but also any protein that binds to the antigen and is pulled out with it. This makes co-ip protocol an ideal technique for studying protein complexes. The main concern when developing a co-ip protocol is to ensure that the lysis, wash, and elution buffers do not denature the proteins. Otherwise the tertiary structure of the proteins will deteriorate and the protein-protein interaction may be altered or completely lost.
Fluorescent nanoparticle is a general term that many people assume means any nanoscale material that produces fluorescence upon excitation with incident light. However, there is actually a thing called a conjugated polymer nanoparticle (CPN) that is very different from the fluorescent dyes (think Alexa fluorophores) that many of us are used to. These CPNs produce higher intensity fluorescence than dyes (up to 1000x brighter!), are stable and much less susceptible to quenching, and don’t contain toxic cadmium like quantum dots. These conjugated polymer fluorescent nanoparticles will likely be at the forefront of future bioimaging methods.
Protein Purification is essential to understanding the structure and function of various proteins. It is also important for the development of clinical biosensors, which rely on stocks of pure protein as detection agents. There are many methods of protein purification, which are discussed below along with how protein purification works, the steps involved, and how to increase yield in your protein purification protocol.
Antibodies are an important part of the immune system. When the body is infected with an antigen, the immune system generates an antibody specific to that antigen. The techniques that are routinely used in biotechnology capitalizes upon this natural immune process. Antibodies are used in many research applications as well as in immunoassays for disease detection. We use the specificity of the antigen/antibody binding for immunoprecipitation and ELISA assays. We use flurophore-conjugated or enzyme-tagged antibodies for labeling molecular targets on individual cells and whole tissue. We use antibody purification to obtain antibodies for biosensors to detect disease. These antibodies, depending on the application, are commonly obtained by antibody purification from humans, rats, rabbits, mice, and chicken.
It is useful, and often necessary, to break apart the cell and separate the cellular organelles into individual fractions for further study. Once the organelles are separated it is easier to identify pathways of disease or basic biochemical functions within the cell. One easy, and relatively gentle (if performed properly) method to do this is via cell centrifugation. This process involves three main steps: homogenization of the cellular extract, differential sedimentation, and density gradient centrifugation. The homogenization step breaks apart the cell membranes and releases the organelles into one big cellular soup. Then, the first step of centrifugation begins with differential sedimentation. This results in a rough separation of organelles. The fractions are further separated into clean fractions by density gradient centrifugation, which uses a material gradient (often sucrose) to help separate the organelles by density during centrifugation.