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Principle: The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents ( i.e. DTT and beta—mercaptoethanol) and metal chelators ( i.e. EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding. Materials and Reagents Bovine Serum Abumin (BSA) (Sigma-Aldrich) Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 ) Methanol Phosphoric acid (H3PO4) Bradford reagent (see Recipes) Equipment Spectrophotometer (Tecan) Whatman #1 paper (Whatman) Procedure ...

Introduction One of the major advantages of cell culture is the ability to manipulate the physicochemical (i.e., temperature, pH, osmotic pressure, O2 and CO2 tension) and the physiological environment (i.e., hormone and nutrient concentrations) in which the cells propagate. With the exception of temperature, the culture environment is controlled by the growth media. While the physiological environment of the culture is not as well defined as its physicochemical environment, a better understanding of the components of serum, the identification of the growth factors necessary for proliferation, and a better appreciation of the microenvironment of cells in culture (i.e., cell-cell interactions, diffusion of gases, interactions with the matrix) now allow the culture of certain cell lines in serum-free media. Adherent vs Suspension Culture: A. Adherent Culture: Appropriate for most cell types, including primary cultures. Requires periodic passaging, but allows easy visu...

The two most common methods of preparing samples for immunoblots are either to lyse the entire cell or tissue mass in sample buffer and load the total cell lysate on the gel or mix any protein sample 1:1 with double-strength sample buffer and load. Preparing total cell lysates: In this procedure, samples of cells or tissues are disrupted in an electrophoresis sample buffer. After the chromosomal DNA is sheared to lower viscosity, the samples are processed and run as usual for particular gel system. In the example given here, the samples are prepared for electrophoresis in a standard Tris/glycinE SDS-Polyacrylamide gel. Measure the volume of sample to be lysed. For tissue culture cells 10 9 cells = 1 ml = 1 gram. For tissues or organs, weighing the sample will be sufficient. 1 gram= 1 ml. Most samples should be boiled for 5 mins. Some antigen will come out of solution under these conditions; if no signal is seen, check different temperature. Soniate the sample to shear t...

Guides for Selecting the Best Primary Antibody   1.  Species   One important factor that should be taken into consideration early in your antibody search is the species of animal in which the primary antibody was generated and whether it will be compatible with your application. If you need to use a conjugated secondary antibody to detect the primary antibody (e.g. when performing immunohistochemistry)  the primary antibody should be from a species as phylogenetically distinct as possible from the species of the sample.   This will prevent cross-reactivity between the secondary antibody and endogenous immunoglobulins in the sample.   2.  The type of antibody matters   Is it better to us a monoclonal or a polyclonal antibody?  For some proteins, you don’t have a say in the matter  you have to go with whatever antibody is available. However, if you do have a choice, choosing the right kind of antibody might increase yo...