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16.6: Micro-report 5- SDS-PAGE and western blot analysis

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    In this report, you will describe the results of the SDS-PAGE and western blots that you used to analyze protein expression in your transformed cells under both repressed and induced conditions. Pay special attention to the degree to which these results confirm or contradict the results of the previous transformation/complementation report. The figure should be labeled in such a way that an experienced scientist is able to understand your results from the figure and legend alone. Many experimental details have been relegated to the M&M section. Lanes should be clearly labeled and the molecular weights of the standards should be included. (The example below is from a different class, where students used SDS-PAGE and western blots to analyze overexpression of yeast proteins expressed from pBG1805. Note that, unlike your experiments, a primary antibody to the HA epitope was used to detect proteins on western blots.)

    Materials and Methods: Provide information about transformed strains, incubation conditions, preparation of cell extracts, SDS-PAGE gels and western blots. Reference published procedures when possible, noting any modifications. Subheadings may be helpful.

    Transformed strains: Include the names of the strains and plasmids that you used to prepare cell extracts.

    Extracts: Include information on the media and incubation times used to manipulate protein overexpression from the strains. Reference the manual for the extraction procedure, noting any modifications.

    SDS-PAGE gels: Provide details about the % acrylamide of the gels, running time and voltage used for electrophoresis.

    Electrophoretic transfer: Include the time and voltage used to transfer proteins from the SDS- PAGE gel to the PVDF membrane. Refer to the manual for other details.

    Western blot: Include the antibodies that you used for the blot and the conditions (time, tem- perature) that you used for each of the incubations. Include the time that you used to detect overexpressed proteins with TMB. (This gives a some sense of the abundance of the protein in your extracts.) You do NOT need to include all the wash steps - just reference the manual.

    Results and Discussion - Begin by discussing the SDS-PAGE gel. The SDS-PAGE gel provides a snapshot of cellular proteins and a rough comparison of protein concentrations in different extracts. The staining intensity of a band reflects its abundance in the extract.

    • How did the total amount of protein compare between induced and non-induced samples? (What might this indicate about the different carbon sources?)
    • Did you see any changes in individual bands on the gel? Is it possible to detect the fusion protein against the background of other proteins in the extract? Recall that cells have thousands of proteins and that a band may consist of more than one protein species.

      The western blot allows you to detect the fusion protein against the background of other cell proteins. Include a table showing predicted and actual sizes of Met or LacZ proteins detected using the western blot technique. The sizes of the proteins are particularly important. (It is probably not possible to get an exact value of the protein sizes, because of the fuzziness of the standards on the western blots. Nonetheless, you should be able to place the proteins within a certain range.) The plasmid-encoded proteins will be larger than the naturally occurring protein because of the epitope tags encoded by the plasmid. Are the observed sizes what you expected?

    Other questions to address in the Discussion:

    • Did you see evidence of induction by the GAL1 promoter on either the gel or blot?
    • How did the western blot results relate to your complementation results? How do these results enrich/weaken your complementation findings?
    • If you did not detect proteins on your blot, propose some explanations. Compare the samples. Do the other samples provide good positive controls for your technique?

    This page titled 16.6: Micro-report 5- SDS-PAGE and western blot analysis is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Clare M. O’Connor.

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