2 replies to this topic

[assembler01]

Once I determine a good amount of lysate to use by dot blotting, how does that translate to amount of protein needed to load per lane? For example, if I determined either 5uL of lysate or 5ug of total protein gave a good signal by dot blot, should I load the same amount into one lane or do you have to scale it up or down since it will separate on the SDS-PAGE? Also, the area of the dot on the dot blot is larger than a typical band on a western blot which makes me wonder if I should scale it up or down.

Thanks.

[neuron]

I have no experience of dot blot but theoretically I think you need to load more amount as you mentioned first. Because in dot blot the signal is cumulative of all the proteins i.e. total protein whereas in WB your protein will be less than the total protein in any case, so less signal.

However only experts who have some practical experience will be able to give you the correct answer

[mdfenko]

neuron has it a little wrong. total protein is of little consequence in a dot blot (other than that the other proteins may mask the protein of interest). if 5ug gave you a good signal and the gel does not spread the bands too wide then you can try 5ug (or you can try a range of protein amounts).