In E. coli prokaryotic expression vector pET32a, does the TrxA tag lead to non-specific binding in WB and ELISA assays?
A TrxA tag is often used to make sure that recombinant proteins fold and dissolve correctly in E. coli expression systems and other studies that use protein expression. Because this tag is not found naturally in human cells, Western blotting (WB) and enzyme-linked immunosorbent assays (ELISAs) might attach it to the wrong cells.
Non-specific binding may occur depending on several circumstances, but this is not guaranteed. The essential details are as follows:
These are called primary and secondary antibodies. If you use non-specific primary or secondary antibodies in Western blotting or enzyme-linked immunosorbent assays (ELISA), they may bind to the TrxA tag or other background proteins instead of the protein of interest. The end effect may be deceiving because of this. If you want to lessen this possibility, you should use verified, highly specific antibodies against your target protein. Non-specific binding could be changed by the type and amount of blocking solution used in WB or ELISA. Non-specific binding might be reduced by testing various blocking agents.
How to Clean: Non-specific binding may be minimized by using appropriate washing procedures to eliminate unbound or weakly bound antibodies.
The Level of Antibody Prevalence: Non-specific binding may occur if too many antibodies exist. Maximize the efficiency of your antibody dilution so that the smallest quantity required for protein detection is used.
General Bead or Plate Locations: If the reactive areas on the ELISA plate are not blocked, non-specific binding might occur. To avoid this from happening, make sure your blocking step is optimized.
Remember, each new protein and tag combination can need tuning to decrease non-specific binding in your WB or ELISA. It’s crucial to validate and control each experiment individually to prevent false findings.