Hi there,

The loading limit is set for optimized band resolution so basically it will depend on the well width and on the gel thickness and size (and also on the origin of the sample: crude extract or purified material). For minigel with 10 well comb it is said that 50µg is the upper limit for a crude protein extract. In your case detection might also be an issue (if you don't get any specific signal with 25µg, it is hard to imagine you'g get something more relevant with 50...)

25-30 micrograms of protein is enough for this purpose,

you may have some other error.

no need to waste your protein sample by exceeding to 50 micrograms.