Is there a more economical protocol to determine gus activity? In my protocol I use 10 mg X-Gluc/10 ml of work solution, can I use fewer X-Gluc? And how many this solution can be reused? Thanks.
When you have strong signals you can lower the [] X-gluc to 0.5 or 0.25 mg/ml. In case the staining solution has still the same colour you can reuse it (within a short time span). Until now I only reused the staining solution once. It will be trial and error to see what you can do for the tissues you work with.
I used to screen transgenic lines with GUS gene expression. There were 50-100 putative transgenic lines. I screened them in a 48-well titration plates (see Fig.1)**. There were many wells where their tissues did not turn blue. There were also some wells where the tissues turn blue. Some lines were showing very strong GUS expression, even their staining solution turning blue. So, as Eric suggested, I only recycled those staining solutions from those wells where no GUS staining were shown. Those recycled staining solution was placed at -20C for next time use. I generally reuse them 1-2 times if their quality maintain. Always add a 'positive control' (prefer derived from a same 'positive' plant) in the fresh and re-used solution to compare and monitor whether the quality of the staining solution has changed.
**Fig.1 was obtained from web.
I have reused it a couple of times. It really depends on the integrity of the components and time of storage.
@ C.E.A. Bastos,
1. As for the question: can the conc. of X-gluc be reduced? It is hard to say. If you are screening putative transgenic lines with unknown GUS expression activity, you might miss out those transgenic lines with low GUS activity if reduced x-gluc conc. is used..
2. How much did your lab pay for the X-gluc? There are some small companies that sell good quality X-gluc with much cheaper price (compared to some big-name companies). You should shop around and do some price comparison.
@ C. E.A. Bastos,
I just went online to check the price of X-gluc.
My lab used to purchase X-gluc from Gold Biotechnology. The quality is pretty good. The price now they are offing is:
(1) 100 mg for $43 (US)
(2) 1 g (1000 mg) for $162 (US) [= 100 mg for $16.2]
In this case, we usually opt for the 1g. There are a couple of people using them in the lab. Bedside, it can be saved at -20C for quite a long time (powder form).
https://www.goldbio.com/category/x-alpha-gal-x-gluc-mug
For Sigma, they are selling X-gluc tablets, each tablet contains 10 mg X-gluc. For 5 tablets, they sell $251. That translates it into 100 mg = $502 (US).
http://www.sigmaaldrich.com/catalog/product/sigma/b8174?lang=en®ion=US
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