Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting
Dialysis and TEV Cut Procedure
The Dialysis and TEV Cut was executed after Wester blotting, which confirmed that the solution contained HRas G12V. The purpose of dialysis and TEV cut was to get a pure/independent HRas G12V without extraneous parts. For the experiment, from the start, HRas G12V was tagged with Histidine to act as an indicator of HRas G12V during Ni-NTA Affinity Chromatography since Histidine binds with Nickel. Therefore TEV protease cut HRas G12Vand the Histidine tagged, leaving pure HRas G12V. At the same time, dialysis was performed to get rid of imidazole. Since imidazole was used to wash separated HRas G12V from the agarose gel, imidazole was in the HRas G12V solution. Imidazole limited TEV protease function, therefore, must be removed. After, another Ni-NTA affinity chromatography was done to separate the TEV protease and the detached histidine tag from the HRas G12V solution. It was possible because TEV proteases have Histidine.
To verify that the experiment was a success (that TEV protease cut the Histidine tag of most of the Histidine tagged HRas G12V) the protein was transferred onto columns for SDS-Page.
The expected result was a distinct line between 20-25 kDa just a little bit below the line shown on the uncut variable since HRas G12V is 21.4kDa. Unexpectedly the SDS-Page results came out as a smudge with several different small lines, instead of a line making it impossible to verify whether or not the solution was composed of pure HRas G12V.
There is no definite cause for the unclear result, however, there are several possibilities.Contaminated Solution:The solution inserted in the SDS-Page columns might have been contaminated with other proteins/contaminants. This explains the numerous small strands shown on the ‘Cut’ column which is supposed to show one distinct line between 20-25kDa that indicates HRas G12V. Such contaminants can be traced back to the protein samples being kept under poor conditions.Band Pattern:The smudged lines shown on the graph can be the natural line.
Since there are so many possible reasons why SDS-Page results came out in a smudge, we decided to verify the transferred membrane by requesting sequencing. It is nearly impossible to find out the exact reason as to why there was a smudge unless a very apparent clue comes out during the process. It was also very likely to fail again because we would repeat the same process without knowing the exact reason. Therefore, we concluded that it would be more time and cost-effective to request our transferred membrane sequencing.
As an alternative solution to verify that the protein solution contains pure HRas G12V, we sent the protein sample to a company that sequenced the given sample through HPLC chromatography. Thankfully the sequenced protein was identical to the sequence of HRas G12V, which verified that the protein was purified HRas G12V that we were looking for.
What We Learned
From this process, we learned that finding an alternate method can sometimes be more effective than holding onto just one type of method. If we only stuck to Western blot and redid the process without sequencing until we got precise results, we would have discarded the wanted protein. Also, we could have spent days figuring out the exact reason why the results came out unclear. Without knowing the failure's specific cause, it is always good to seek other verification methods.