The isolation of protein from cultured cells is important for both analysis and production. Analytically, proteins are measured by activity as with enzymes or biochemically through separation techniques (i.e., chromatography and electrophoresis) or immunoassays (i.e., antigenicity). Activity assays can take on several forms, such as protein activity assays and cell based assays where the protein affects cellular behavior, as with a mitogen. In both cases, the proteins must be functionally active and harvested in a non-denaturing manner. Immunoassays, on-the-other-hand, involve an antigen-antibody interaction which does not require functionally active antigen (i.e., the protein). Consequently, methods for harvesting protein for immunoassays may include denaturing conditions. This is espeically true of proteins analyzed by Western blotting, a process which first involves separating the proteins by SDS-polyacrylamide gel electrophoresis (PAGE).
A protein activity assay is a measurement of the protein's biological activity as it correlates to protein purity and concentration. Of course, the assumption made here is that the protein has a biological activity that can be measured. The protein activity assay is to be distinguished from the protein assay or assay for total protein concentration. The assay for total protein concentration simply quantitates the mass of protein present in a sample, based on the chemical or physical properties of the protein.
An example of a protein activity assay is the enzyme assay for α-galactosidase. The assay is based on the colorimetric determination of p-nitrophenol, which is released from the synthetic substrate, p-nitrophenyl-α-D-galactoside, by α-galactosidase. An enzyme assay, therefore, is a specific type of protein activity assay.
Biochemical reactions are defined by the rate of the reaction, where reaction rate is defined as the disappearance of substrate or, the appearance of product over, time. The biological function of any enzyme is to accelerate the rate of the associated biochemical reaction. An enzyme assay is a special type of protein activity assay where the increase in reaction rate catalyzed by the enzyme is measured and quantified. This measurement, reflecting the reaction rate increase, correlates with the concentration of the particular enzyme. Simplified, the faster a substrate is consumed or a product accumulates, the greater the concentration of the enzyme.
Enzyme reaction rates increase by a factor of 1.5 to 2.2 for every 10°C increase in temperature. The optimum temperature for an enzyme is actually dependent upon the assay method. At high temperature, some enzyme is lost to denaturation, so increasing temperature leads to decreasing product formation. The shorter the incubation time, the higher the apparent optimum temperature. It is useful to establish a temperature zone in which the enzyme is stable and has high activity. In cell based assays, factors such as temperature distribution within an incubator can have an important impact on the rate of reactions. For instance, some incubators may have elevated temperatures near heated doors. This can cause drastic variations in enzyme rates during assays between cells in the front of an incubator as compared to those in the rear.
A number of different lysis buffers can be used to release proteins from cells depending on the application and the cell type. For activity assays, the primary concern is to release an acitive biomolecule. While for immunoassays, the primary considerations are efficient release of the protein antigens and recognition by the antibody after isolation (structural integrity). Variables that affect both of these parameters are salt concentration, type of detergent, divalent cations, and pH. Salt concentrations between 0 and 1 M, non-ionic detergent concentrations between 0.1 and 2.0 %, ionic detergents between concentrations of 0.1 to 0.5 %, EDTA concentrations between 0 and 5 mM, and pH between 6 and 9 have been used successfully in a variety of different protocols. Also, to avoid proteolysis, EDTA, aprotinin, and PMSF (phenylmethylsulfonyl chloride) are often added to inactivate endogenous proteases.
Preparation of protein solutions for use in activity assays, electrophoretic analysis, or immune reactions (immunoprecipitations or Western blots) may be accomplished in a number of ways. For many cells grown in culture, the most useful method of lysis is treatment with detergents. Cells can also be disrupted by devices generating physical shearing forces such as blenders, ultrasonicators, and homogenizers. These are particularly useful for large volumes. Freeze-thaw lysis should be avoided as it is generally found to lead to extensive degradation of the proteins, presumably by proteolysis. Denaturing lysis is useful if one knows beforehand that the antibody will recognize a denatured protein or that the protein will refold correctly after removal or dilution of the denaturant. Detergent lysis is routinely used as the first choice for preparing immunoprecipitations.
| RIPA Lysis Buffer | NP-40 Lysis Buffer |
|
150 mM NaCl 1.0 % (v/v) NP-40 0.5% (w/v) sodium deoxycholate 0.1 % (v/v) SDS 50 mM Tris pH 7.5 |
150 mM NaCl 1.0 % NP-40 50 mM Tris pH 8.0 |