blg888.docx - Introduction Western Blotting is a powerful...

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Introduction: Western Blotting is a powerful technique for the detection of low abundance proteins that forms an exact copy of SDS (Sodium dodecyle sulfate) polyacrylamide gel, then, the primary/secondary antibodies are added with an enzyme conjugate to develop the color of protein bands for detection of protein (Kurein & Scofield, 2006). Western blotting is used to purify proteins and monospecific antibodies, also used for the detecting proteins and enzymes with the use of different ligands or antibodies (Rosenbaum et al., 1989). In Western blotting, Proteins are divided as per their molecular weight and type via gel electrophoresis, the obtained results are transferred to a membrane for visualization of bands (Mahmood & Yang, 2012). The protein specific antibodies are used on the membrane for the detection of a specific protein and the non-required antibodies are washed off the membrane leaving the protein of interest visible for detection (Mahmood & Yang, 2012). SDS-PAGE separate the protein bands then, protein specific stains followed by distaining with deionized water results in appearance visible of protein bands (Pal, Godbole, & Sharma, 2004). The purpose of this experiment is to detect the -Galactosidase protein using the technique of western blot. Three different techniques will be used in this experiment, E.coli strains EMG 26 K-12 lac - (i + z - y + ) and EMG 9 K-12 lac - (i - z + y + ) will be used to isolate the cellular proteins, SDS-PAGE will be used to produce protein profile based on molecular weight. Half of the gel will be stained with Coomasie Blue for appearance of all proteins and, the other half will be transferred on nitrocellulose filter for the observance of -galactosidase with the use of an anti- -galactosidase monoclonal antibody followed by anti-IgG alkaline phosphatase conjugate.
Coomassie Blue staining (CBS) is the most commonly used staining method for the detection of proteins separated by SDS-PAGE. There are several other staining techniques such as fluorescence or silver staining but CBS is a low-cost, simple, compatible and highly effective staining method ( Májek et al ., 2013) . The ionic interactions between dye sulfonic acid groups and positive protein amine groups results in binding of coomassie blue with proteins, sometimes, Van der Waals forces are involved as well ( Májek et al ., 2013) . B-Galactosidase detection with Nitrocellulose is a method specific to this protein as compare to Commassie Blue which detects almost all protiens. In this detection system, antibody binds to protein and the complex binds an antibody conjugate which then recognize th covalently linked alkaline phosphate lgG antibody and reacts with BCIP (5-bromo-4-chloro-3 indolyl

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