We use a variety of in vitro biochemical assays in the laboratory. One of these is the in vitro splicing assay, which uses a 32P-labeled pre-mRNA template to assay splicing activity of nuclear extracts under normal conditions and following the addition and/or removal of specific proteins. Shown here is an example of an in vitro splicing assay, separated by SDS-PAGE and detected by autoradiography. The pre-mRNA lane shows the input (labeled template), while the Control lane shows the splicing intermediates and products. The cartoons below define each of these:
In this particular experiment, while addition of GST alone has no effect on splicing (lane 3), we showed that addition of a dominant-negative mutant of NIPP1 (PP1 binding ability severely reduced) causes a buildup of intermediates, indicating an adverse effect on the catalytic steps (see lanes 4-11).
This gel was taken from the following publication: "Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors". Trinkle-Mulcahy, L., Ajuh, P., Prescott, A., Claverie-Martin, F., Cohen, Stanley, Lamond, A.I. and Cohen, P. (1999) Journal of Cell Science 112, pp 157-168.

Another common biochemical assay performed in the laboratory is the in vitro phosphatase assay. This is based on work published by Philip Cohen and co-workers, and a description and citation can be found in the NIPP1 publication listed above, which is also the source of the sample figure shown here. In this case, the phosphatase activity of purified PP1 is measured as increasing amounts of GST-NIPP1 (open circles) or GST alone (closed circles) are added. It can be clearly seen that NIPP1 is a potent inhibitor of PP1 against the phosphorylase a substrate used in this assay. The assay can also be used to measure total phosphatase activity in cell lysates, and can be adapted to use different substrates.

Both of these assays can be found on our Protocols page.