Western Blot has been a powerful tool in life science labs for over thirty years. One of the encumbrances of this method is that it is labor and time intensive. In our lab, shortened incubation times are acceptable for identity testing, however long incubation times are still standard when detecting low amounts of antigen or when using low affinity antibodies. We evaluated the Millipore SNAP i.d™ for use in routine western blot analysis as well as for researching new proteins whose properties were not as well known. The SNAP i.d.™ uses vacuum assisted diffusion to greatly decrease incubation times. A variety of samples including recombinant proteins and antibody fragments were optimized for both methods. Proteins were transferred using the Invitrogen i‐Blot®. Direct dual‐development analysis, staining the blot with Sypro® Ruby prior to antibody exposure was used to differentiate closely migrating proteins from the protein of interest. Commercially available kits were utilized to develop the blots. This study indicated that the SNAP i.d.™ is suitable for a variety of applications of the Western blot technique, although substantial method development may be required for unfamiliar proteins and specific applications. The system was useful for quickly identifying epitopes in specific recombinant degradation products, aiding in the identification of the specific cleavage sites and detecting low level contaminants.
Measuring the concentration of peptides can be challenging. Amino acid sequence, solubility and the folding of the peptide can influence measurement. A280 measurement requires that chromophores trytophan and tyrosine be in the sequence. Lyophilized peptides contain water and salts so concentration determined by weight alone is imprecise. Amino acid analysis is the default method for measuring peptide concentration, but it is very labor intensive. We evaluated two microtiter plate methods for potential use in peptide quantitation. Pierce BCA Protein Assay (bicinchoninic acid) combines the reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the selective colorimetric detection of the (Cu1+) by bicinchoninic acid. Peptides of three or more residues form the reaction, which is measured at 562 nm. LavaPep™ Protein & Peptide quantification kit depends on a small, naturally‐occurring fluorescent compound, epicocconone, that reversibly binds to lysine, arginine, and histidine residues to yield an intensely red‐fluorescent product (Ex/Em: 540/620 nm). Various peptides were used to generate standard curves to characterize the affect of standards used on the concentration. Results presented will demonstrate the relative robustness, accuracy and precision of the two methods.
The conformational stability of proteins can be impacted by the oxidation/reduction (Redox) of disulfide bonds. Redox sensitivity is often more pronounced for proteins that are coated onto a magnetic particle solid support due to the increased presence of heavy metal ions. In the case of immunoassay performance, a reducing environment must be maintained in solution for proper protein activity over the shelf life of the product. Diluents were designed with a number of metal chelators (EDTA, DTPA) and various levels of dithiothreitol (DTT) and glutathione to determine the impact of metal chelator on the stability of the reducing environment. A modified microtiter method was developed utilizing the Ellman's reagent (5,5′‐dithiobis‐(2‐nitrobenzoic acid) or DTNB) to provide a simple, robust and accurate quantitation of total sulfhydryl (SH) concentration. Using an accelerated stability model that incorporated various SH concentrations, temperatures, and storage times, the remaining SH concentrations were measured to determine a formulation that resulted in the most stable reducing environment. A formulation containing the chelator DTPA was shown to be the most successful and approximately doubled the stability of the diluents’ reducing environment in comparison to EDTA as measured per the model.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.