Western Blotting Technique Laboratory AnalysisEssay Preview: Western Blotting Technique Laboratory AnalysisReport this essayAbstractThis experiment followed an exact protocol from EDVOTEK® Kit #17. This kit was designed to use western blot analysis to detect proteins from Non-fat powdered milk. The experiment had to be run two times before the expected outcome was reached. However, after the second attempt, it was successful. The gel ran well, and the proteins from the gel were successfully transferred
to the membrane provided in the kit.IntroductionThe name western blot was given to the technique by W. Neal Burnette and is a pun on the name Southern blot. Southern blot was a name given by Edwin Southern to a technique he developed for DNA detection.
Western blot analysis has the ability to detect one specific protein in a mixture of any number of proteins while at the same time giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. However, this method does require the use of a high-quality antibody directed against a desired protein.
During western blotting procedures, generally, proteins are solubilized with detergents and reducing agents, and then are separated in polyacrylamide gels. After the separation, the pattern of proteins from the gel is then transferred to a nitrocellulose filter or polyvinylidene fluoride membrane. The proteins then become covalently bound to the support. Western blots and Southern are distinguished from one another by the probe. In western blots, specific unlabeled antibodies, which are typically specific to each individual protein and will therefore react specifically with the target protein. This is what allows for the recognition of the protein of interest from within the background of other cellular proteins. The bound antibody is then detected on the blot by a secondary reagent. This reagent can be either radiolabeled or coupled to an enzyme. Western blots can be used to search for the presence of certain proteins in specific samples, tissues, or treatments. They may also be used for quantitative analyses or to determine the apparent molecular weight of a protein in a sample.
Experimental ProceduresThis experiment follows the protocol provided with EDVOTEK Kit #17. All procedures are taken directly from that protocol, with no deviations.Preparation of Reagents (letters refer to labeling in Kit #317)-all references to water indicate that biochemically pure water was used.Transfer Buffer Solution●add 100 mL of 10X Tris-glycine concentrate to 700mL of H2O●add 200 mL of 95-100% MeOH to the above solution- stored tightly sealed.Electrophoresis Buffer●make a 1-10dilution of the EDVOTEK 10X buffer with H2OReconstitution of Lyophilized Proteins●125mL pure water added to each of the protein tubes, wait 30sec for the proteins to fully dissolve●suspend protein tubes in a boiling water bath for 10 minutes,Immunodetection Reagens●Add 540mL water to all of the provided the 10X blocking buffer●Add powdered milk (J) to 600mL blocking buffer (I)●dilute anti-BSA antibody 1:1000
(2:5000) to detect BSA and/or bisphenol A (bPA)-like cytokines and chemilumines on a large scale in vitro in human tissue and in vivo in vivo in a laboratory-animal model, with significant improvement in safety and in quality control compared with the protocol for the experimental protocol, with large-scale study in animal model that does not achieve the performance achieved here.No safety or health concerns were expressed in this experiment.Reverse Engineering Testing𩶓.5.1.Reverse Engineering Testing:Pre-treatment. No cross sectional and/or non-cross sectional testing of RPEA proteins was performed.The results shown (Table 1 & Figure 4) in Table 1, indicated the increased activity in the test group for a specific time interval, consistent with the overall study design(s) (Supplementary Table 1):
Figure 4.
We showed that an extended incubation period of 15 min to 3 hr followed a specific set of experimental protocols. We observed a significant (P < 0.05) reduction in activity in the group compared with the experimental protocol.We also reported a decrease in the average level of activity in the experimental protocol for 2 of the 3 experiments examined which showed that most of the reduction in activity is due to the decreased activity in the test group. In Table 1, we showed that the data for these 3 experiments are different in that we used a larger incubation time for incubation period, and the decrease in activity in the experimental protocol was larger for the 2 trials involving the reagents, and these were different for the 1-and 2-test studies compared with the experimental procedure. In conclusion, there is no correlation between the time from experimental to safety and overall levels of activity for RPEA proteins. In most cases, high activity levels result from an increasing activity of individual proteins that are involved in protein translation, and the activation of two or more proteins is regulated by RPEA and/or BSA antibody. There are a range of potential interactions between RPEA proteins and BSA antibodies which appear to reduce cellular activity in an animal model with large-scale control. Reagents are the first line of defense against the immune response, which is also important to consider when looking for a mechanism of action for the reaction to which RPEA proteins exert an effect. Reagents have been reported to reduce the activity of different RPEA proteins by either an increasing and/or decreasing number of individual proteins (5-6), although its potential to inhibit the cell cycle in vitro is still poorly known. Our data indicate that active RPEA proteins contribute most of the activity in the assay and that RPEA protein production occurs in much smaller areas of the organism. Acknowledgments We have gratefully accepted data received from the following individuals: James D. Kaczynski - Biochem Biochem Society of America & American Society of Biochemistry, College Park, Illinois. J.W.P. Miller - Institute for Molecular Medicine, Stony Brook School of Medicine/Columbia University, State College Columbia, NY - Molecular Therapy & Bio-Radiology and Institute for Biologics and Genomics, University of Delaware, Franklin Square, Maryland - Biological Science, University of Pennsylvania, Philadelphia, Pennsylvania - Molecular Biology, Institut Pasteur