Abstract
Epicocconone, a naturally occurring fluorophore that alters its fluorescent character upon binding to proteins is the basis for a number of new labeling technologies ranging from liquid protein quantitation to gel stains and cell stains. LavaPurple is a highly sensitive total protein gel stain that may be used as a stain for nitrocellulose and PVDF blots. The high sensitivity of the stain, coupled with the unique reversible covalent binding of epicocconone, make it an ideal blot stain as it is easily removed by pH change thus not interfering with downstream immuno-staining, mass spectrometry or Edman sequencing. Aim: This study measures the sensitivity of LavaPurple as a blot stain on both nitrocellulose and PVDF membranes, as well as the compatibility of the stain with immuno-staining. Method: a dilution series of Low molecular weight SDS-PAGE calibration markers (GE Healthcare) were separated by electrophoresis and electro-blotted onto either nitrocellulose or PVDF. The blots were then stained with LavaPurple and image analysis performed to determine sensitivity and linearity of the stain. Cell lysate from ovarian cancer cells and rat fibroblasts were separated by 2D GE and blotted onto both nitrocellulose and PVDF membranes before staining with LavaPurple. The blots were then used for immuno-staining and the efficiency of visualization compared with and without total protein pre-staining. Results: LavaPurple was able to visualise proteins on blots at less than 250pg/band loaded on a 1D gel and did not inhibit subsequent antibody binding to target proteins on either 1D or 2D blots. Furthermore the stain could be easily removed from the blot for subsequent staining with other fluorophores. Conclusions: LavaPurple is a highly sensitive stain that is suitable for total protein visualization on blots either for spot removal or prior to immuno-staining.