Question

What result would you obtain if you carried out the incubation with an anti-GFP antibody instead of anti-RFP antibody as the primary antibody? Explain why.

What result would you obtain if you switched the antibodies? Meaning the primary antibody was used as the secondary antibody and vice versa. Explain what is happening on the blot and what is detected.

Answer 1

Fluorescent proteins are unique, large , ~25 KD proteins with the property of bioluminescence. The first fluorescent protein to be discovered was the green fluorescent protein (GFP) naturally present in the jellyfish Aequoria victoria way back in 1962. Most of the other fluorescent proteins (emitting different colours) including the red fluorescent protein or RFP are derived from GFP through mutations. However, there also exist naturally occuring RFP such as the DsRED2 RFP obtained from the red coral, Discoma sp.

The basic structure of all the fluorescent proteins are the same. It constitutes of an outer rigid, beta-barrel fold comprising of 11 beta sheets and an internal, central alpha-helix. The chromophore element, that is responsible for the property of fluorescence actually comprises of only four conserved amino acid residues located at the centre of the beta-barrel. These amino acids are usually highly polar. The fluorescence of a FP depends highly on the chemical environment surrounding these amino acids.

When GFP is mutated to give rise to different coloured fluorescent proteins, specific amino acid residues within the beta-barrel are usually mutated that alter the fluorescent and other spectral properties of the protein.

If an anti-GFP antibody is incubated instead of an anti-RFP primary antibody, there could be two results.

1. If the anti-GFP antibody was raised against the common epitopes (antigenic amino acid residues) of GFP and RFP, then even the anti-GFP antibody will pick up signal from incubating with RFP protein and we will see the RFP band on the blot after performing western blot.

2, If the anti-GFP antibody was raised against the specific amino acids that were mutated in GFP to give rise to RFP, then the anti-GFP antibody can't recognise the RFP protein, since, those amino acids are no longer present in RFP. In that case, there will be no signal on the blot.

If we swap the antibodies, that is, if we substitute primary with secondary antibody and vice-versa, then either we will see no signal or we will see a lot of non-specific signals on the blot.

The primary antibody usually targets a specific site in our protein of interest. Secondary antibody targets the primary antibody. Further, secondary antibody is usually conjugated with a fluorophore. So, if we first add the secondary antibody, there will be no specific binding. If any signal appears, it will be non-specific in nature. And when we add the primary antibody, there will definitely be specific binding to the target. But because, we will not add secondary antibody again, the binding will not be reflected as any band on the blot, since primary antibody is not conjugated with any fluorophore.