I'm expressing 3XFLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK, ~2.9 kda) using in vitro translation system and trying to detect this peptide using western blot (1st Ab = Monoclonal ANTI-FLAG® M2 antibody produced in mouse).
I will use tricine-SDS-PAGE and blot peptide on PVDF or positively charged nylon membrane.
However, in the literature of 'Anti-FLAG® M2 Magnetic Beads' Figure. 4 (https://www.sigmaaldrich.com/KR/en/technical-documents/technical-article/protein-biology/protein-purification/anti-flag-m2-magnetic-beads, and image below), when 3x FLAG peptide was immunoblotted, then It looks peptide cannot be detected by Anti FLAG antibody.
So, I think that transfer condition can be problematic but not sure.
Does anyone see 3xFLAG peptide detected by western blot or can it be detected by immunoblotting?
Also, advice on the experimental process is always welcome.
I've never tried this but could it be that the pore size of the PVDF membrane (typically 0.45 um) is just not binding such a small 3xFLAG peptide and during transfer its not adsorbing to the hydrophobic surface and instead blowing through during transfer? You would probably have to test transfer conditions and probably use 0.22 um membrane so that the pore size is tighter.
I would suggest is try to run the peptide on Tricine SDS PAGE and then perform an hour of crosslinking with formaldehyde and then perform coomassie stain (G-250) to see them on gel first. These peptides are low mol weight so they can easily escape from the pore of gel and hence crosslinking will trap them. Off-course run with a low mol weight protein ladder. Once you see your desired peptide which means your peptide is getting expressed and you can then perform western blot.
For western transfer of low weight peptides, I usually separate them first on Tricine SDS PAGE and then perform transfer for 40 mins at 100 V on 0.45 um membrane. After 40 mins, I usually can see proper transfer of all my low weight ladder. For staining I usually take a little high con. of primary antibody made in 5% BSA. It has worked perfectly well for all my hydrophobic peptides.
- Badges
- Science method