Yes. Everything should be the same for the standards as for the samples. That also includes the buffer that the samples were in, if there is any possibility that buffer components will affect the signal (in the Lowry assay, this is the case for reducing agents, for example).

Agree with Adam. All the parameters including the volume need to be the same for both standards and unknown sample.

Hello Rakesh

Whenever unknown samples are to be run along with the standards the total volume of both have to be the same in order to obtain correct results. Uniformity must be maintained throughout.