beta-galactosidase half-life - (Apr/25/2001 )

I'm doing beta-galactosidase assays right now in E.coli with an alpha-complementing transcriptional fusion vector. The promoter is fused (transcriptionally) with the alpha comp. fragment, and when expressed in a lacZdeltaM13 strain, turns blue. Anyway, does anyone know the half life of (either of) these fragments? I don't really feel like doing a northern blot to asses message stability or a western to see how fast it dissappears, but this has relevance for the levels of beta-gal activity (e.g. is a plateau really a steady-state level of expression or just what was already there?).Thanks, e

I do not know the answer to your question, persay, but I have been working with B-gals for a while. One thing that many people do frequently is to grow a culture ON, dilute it the next morning, and then induce and assay that culture. This can allow your protein (this will depend upon the protein--yours may be processed quickly, so this may not apply) to build up and saturate your assay, in which case your results will not be accurate or consistant.Try this: Day 1--Set up a 5 ml culture ON. Day 2--AM dilute that culture 50 ul:5 ml. PM dilute again. Day 3--dilute once more to a working density and then grow, induce and assay. It's time consuming, but it may be worth it. The multiple passages maintain a basal level of your protein. Good luck.