Cell Biology Laboratory #12- Immunodetection (Western Blots) Introduction Goal: EGF binds to an RTK, or receptor tyrosine kinase, and activation of this enzymelinked receptor results in the phosphorylation of tyrosine residues on intracellular proteins. In this laboratory, SDS-polyacrylamide gel electrophoresis and Western Blotting were used to identify the proteins that possess phosphotyrosines in response to EGF treatment. The proteins were visualized through the cleavage of a luminescent substrate by Horse Radish Peroxidase (HRP), an enzyme associated with avidin and biotinylated secondary anti-mouse IgG antibody. Identification of the phosphotyrosine proteins determined the proteins affected by EGF signaling, demonstrating the use for the Western Blotting technique in research for protein identification. Materials and Method Human epithelial cells (A431) were prepared into two cell homogenates, one with EGF and the other without EGF, and then treated with SDS sample buffer, a phosphatase inhibitor, and a protease inhibitor. These two homogenates were run on a gel with a prestained MW marker and a biotinylated MW marker. After the gels were done running, they were blocked with milk and soaked in primary mouse monoclonal antibody. This was followed with a secondary biotinylated anti-mouse IgG antibody, then avidin/HRP conjugate, and finally a luminescent substrate. The gels were visualized after incubation with the luminescent substrate and photographed for analysis.1 Results 1. The pictures of the experimental blot and the two negative control blots are attached. 2. The graph of Rf vs. Log of Molecular Weight is attached. Using the data, a line of best fit was calculated and was found to be Rf= -0.906(log of MW) + 5.09. The Rf value for the predominantly labeled protein in the experimental blot was found to be 0.25. This was substituted into the equation above and thus the log of MW for this protein was determined to be 5.3416. After solving for the molecular weight through the inverse of the log function, the molecular weight was determined to be 10(5.3416) or approximately 219, 584 daltons. 3. Based on experimental blot, EGF appears to either affect one protein with a molecular weight of 219, 584 daltons or different proteins with that same molecular weight in the cell. This appears to be the case because only one band 1 “Laboratory 12: Immunodectection”. This experimental handout is used by Dr.Homer for the Cell Biology Laboratory course at Johns Hopkins University. appeared in the experimental conditions, indicating that the EGF receptors phosphorylate proteins with molecular weight 219, 584 daltons. It is more likely that this is one protein since it is improbable that numerous proteins in the cell have the same molecular weight with their variations in length and amino acid sequence. 4. The first negative control without primary antibody confirmed that the secondary antibody binds only to the primary antibody since there was no bands present after the secondary antibody was applied. The lack of bands indicates no secondary antibody binding. The second negative control confirms that any band visualization is a result of secondary antibody binding. There were no bands present when only primary antibodies were applied to the gel. This confirms that the bands visualized in the experimental condition was not be caused by a faulty primary antibody. 5. The drawing of the pancreatic acinar and islet cells is attached. 6. Towbin, H., Staehelin, T. & Gordon, J. “Electrophoretic Transfer of Proteins From Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications.” Proc. Natl. Acad. Sci. U.S.A. 76, no. 9, pp. 4350-4354, Sept. 1979 a. Towbin and Gordon compare their new technique to the Southern technique use to analyze DNA. b. The experimental proteins used in this paper are Escherichia coli ribosomal proteins L7 and L12. c. The sine qua non needed to visualize a specific protein on a Western blot is a reporter molecule (radioactive label, fluorescence label, or enzyeme) linked to either the primary or secondary antibody. Conclusion The results of this experiment indicates that EGF appears to either affect one protein type with a molecular weight of 219, 584 daltons or different proteins with that same molecular weight in the cell. This appears to be the case because only one band appeared in the experimental conditions, indicating that the EGF receptors phosphorylate proteins with molecular weight 219, 584 daltons. It is more likely that this is one protein since it is improbable that numerous proteins in the cell have the same molecular weight with their variations in length and amino acid sequence. The two negative controls, one without primary antibody and the other without secondary antibody, confirmed that band visualization resulted as a result of proper antibody binding. The first negative control, without primary antibody, confirmed that the secondary antibody only binds to the primary antibody since there was no bands present after the secondary antibody was applied. The lack of bands indicates no secondary antibody binding. The second negative control confirms that any band visualization is a result of secondary antibody binding. There were no bands present when only primary antibodies were applied to the gel. This confirms that the bands visualized in the experimental condition was not be caused by a faulty the primary antibody. This experiment demonstrated the use for Western Blotting in identifying proteins of interest, such as phosphotyrosine proteins in this experiment. Identification of the protein in this lab identified the protein affected by EGF treatment and other proteins can be similarly identified using Western Blotting. Possible sources of error include not soaking the gels in either the primary or secondary antibody long enough. This would prevent the antibodies from binding on all available or antigen-binding sites either on the protein, in the case of the primary antibody, or on the primary antibody, in the case of the secondary antibody. This would prevent band visualization on the gel and yield inaccurate results, as not all proteins actually affected with EGF treatment would be accounted for.