• My NEB
  • Print
  • PDF
  • Analysis of Synthesized Protein using PURExpress (E6800)

    Introduction

    After in vitro transcription/translation, reactions can be analyzed by SDS-PAGE followed by staining with Coomassie (see Figure 1 on the main product page), silver or other dye, western blotting or autoradiography (for labeled proteins, see Figure 2 on the main product page). PURExpress reactions are amenable to direct analysis; there is no need to precipitate the proteins by acetone, TCA or ethanol prior to SDS-PAGE. Alternatively, if the target protein has enzymatic activity, the reaction can be used directly in the enzymatic assay provided the reaction mixture components do not interfere with the assay.

    The yield of the target protein will vary. On average, we observe between 10–200 μg/ml, which translates to 250–5000 ng/25 μl reaction volume. It is useful to run a portion of the reaction on a protein gel and compare the banding pattern to a control reaction with no template DNA. The target protein is usually observed as a unique band, not present in the control reaction. Sometimes, the target has the same apparent MW as one of the endogenous proteins. In these cases, the target protein will enhance or “darken” the co-migrating band. We recommend loading 2.5 μl of a reaction onto a mini protein gel and resolving by SDS-PAGE. Briefly, mix 2.5 μl of a PURExpress reaction with SDS gel loading buffer and H2O so the total volume is 10–15 μl. Heat the sample for 2–3 minutes at 90–100°C to denature the proteins and load the sample onto a protein gel. Run the gel according to the manufacturers recommendations and stain with Coomassie Blue or other stain as directed. Proteins below 16 kDa will migrate with the majority of ribosomal proteins at the bottom of the gel.

    To improve resolution in this MW range, we recommend filtering the reaction with a Amicon Ultracel 0.5 ml-100K to remove high MW ribosomes (see figure 3 and figure 4 on product page ) and running the flow-through (permeate) on the SDS gel.

    If the reactions will be visualized by autoradiography (for 35S-met labeled proteins) or by western blotting (for target proteins recognized by an available antibody) the amounts of reaction needed will vary and usually be less than the 2.5 μl used for Coomassie stained gels. Aliquots between 0.5–2.5 μl should be sufficient depending on the efficiency of the labeling, age of the label, or quality of the antibody. Again, we note that in vitro protein synthesis reactions produced by PURExpress can be directly loaded onto SDS-PAGE gels with no need for acetone precipitation and clean-up.