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The objective of this laboratory is to determine from one of the two cultures of E.coli BL21 cells that was induced with isopropyl B-D-1-thiogalactopyranoside (IPTG) to express gene glutathione-S-transferase (GST). The two cultures were transformed with pGEX-2T plasmids because it allows for replication within bacterial cells and expression of a gene of interest. To analyze the proteins two common methods were used SDS-PAGE and Western blot (figure 1 and 3). SDS-PAGE allows to see all of the protein in sample while Western Blot allows to identify specific protein within a mixture of proteins. Our results show that culture A was induced with IPTG to express GST protein while culture B was not induced with IPTG, thus it did not express GST protein.
SDS-PAGE gel can show all of the proteins present in samples. Sodium dodecyl sulfate (SDS) unfolds protein and coats them with negative charge which then are electrophoresed from top (-) to bottom (+) in polyacrylamide gel in parallel lanes. To reveal the proteins they were stained by Coomassie blue in this lab. The results of SDS-PAGE shows that crude A in lane 3 has thick dark spot among the smears of proteins that migrated 46 mm in distance and has size of 23 kD. In lane 8 pure A has one band that migrated 47 mm in distance and has size of 21 kD. The sizes of protein in crude A and pure A was found using semi log plot which estimates size of protein in kD based on distance of migration in mm. Crude B has smears of protein bands and pure B did not have any bands. Based on this information we can infer that culture A was induced by IPTG to express GST protein because sizes of protein in crude A and pure A were close to the size of GST protein which is 23 to 27 kD (figure 1 and 2).
To determine identity of specific protein like GST, Western Blot analysis was done in which the membrane is incubated with primary and secondary antibodies and a color substrate which turns purple when cleaved by secondary antibody later attaches to specific protein of interest which is then color labeled on gel to be easily seen. The results of Western Blot Image shows one purple band in lane 3 of crude A which migrated 47 mm in distance and has size of 24 kD and lane 8 also has purple band which represent pure A which migrated 48 mm in distance and has size of 22 kD. The sizes of protein in crude A and pure A was found using semi log plot which estimates size of protein in kD based on distance of migration in mm. This data confirms that GST protein was expressed in crude A and Pure A because of its size and color which are very close to GST proteins size and color. Crude B in lane 5 and pure B in lane 10 did not have any bands because it was not induced with IPTG to express GST protein. (Figure 3 and 4). This lab was done successfully and there were no source of errors made. Two methods of protein analysis SDS-PAGE and Western Blot Image are very useful because they can be used to detect proteins of interest on a gel in presence of many other proteins and also allows to determine sizes of the proteins.