See lane 3 (flow through fraction after binding of GST-protein to the affinity c
ID: 178415 • Letter: S
Question
See lane 3 (flow through fraction after binding of GST-protein to the affinity column) in Fig. 2. Explain why do you see distinct and abundant protein band at around 65kD which is not present in lane 1, but abundant in all other lanes.
Figure 2. Proteins separated on SDS-PAGE from different fraction during GST-tagged protein expression, extraction and purification. Lane 1 – total protein extracted from E. coli prior IPTG induction; lane 2 – Total protein extract from E. coli after GST-tagged protein induction with IPTG; lane 3 –flow through fraction after running IPTG induced total E.coli protein extract through GSH-agarose column; lane 4 – 1st elution fraction from GSH-agarose column; lane 5 – 2nd elution fraction from GSH-agarose column. (Note: wash fractions were not analysed on this SDS-PAGE)
kD 116 66.2 45.0 35.0Explanation / Answer
We see distinct and abundant protein band at around 65kD in lane 3 which represents a flow through fraction after running IPTG induced total E.coli protein extract through GSH-agarose column. Since we know that; Glutathione S-transferase (GST) is a 211 amino acid protein (26kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins (Thermofisher). When we are seeing a distinct abundant protein band in lane 3, which is an experimental group having treatment with IPTG it can be inferred that IPTG is inducing the over expression gene responsible for the increased levels of the protein of 65kD in lane 3 .