I have constructed a line of a stress inducible gene having native promoter::CDS:: GFP in Arabidopsis. The molecular weight of protein of interest+ GFP is 51kda. However, while performing western blot, the anti-GFP antibody (1:10,000 dilution) also binds to rubisco which also around 50kda according to the ladder. How can i detect the band of my protein of interest? I have also tried to increase washes after antibody incubation.
Native promoter always is difficult. In my experience with ab290 it was important to block for extended time (we do PBS + 5% milk buffer (over night or at least 3-4h with changing buffer a few times). Solve ab290 in PBS + 5% milk also, actually we use 1:2500 dilution of the primary; 1:10000 seems very diluted. You can reuse the ab several times (store at -20) even if in milkbuffer (can add azide but even without it last for a while.). Use a tissue with the highest possible expression (maybe you need to cut away unimportant parts of plant material, let's say it is more expressed in young tissues->cut away everything older; this gives you more signal and maybe you can detect it vs a negative control even if there is some background). If you can use tissue with less chloroplasts it might also help and using a very stringent extraction. If you have a nuclear protein you could try to go for nuclear isolation to get rid of chloroplasts. If you want to shary your potocol, happy to have a look.
If your GFP-tagged protein is expressed in roots that is a good source tissue with little rubisco. You'd grow your plants in a hydroponic system or synthetic solid medium (not soil).
Alternatively, sometimes a different antibody batch or source can help. Some companies will send you a small test sample if you ask. It can be challenging to avoid all cross-reactivity for plant samples.
I have similar problem with GFP ab290 in Arabidopsis seedlings, where I observed 2-3 non-specific bands. In the end I switch to Roche GFP ab, work very well for me.
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