GE Healthcare’s AuroDye™ Forte Colloidal Gold reagent is a stabilized gold sol (i.e. suspension), adjusted to a pH of about 3. At this low pH, negatively charged gold particles bind very selectively to proteins by hydrophobic and ionic interactions. Due to the optical characteristics of the gold particles, AuroDye™ Forte stains transferred proteins dark red. The sensitivity is at least comparable to the silver staining of proteins in polyacrylamide gels. It has been shown to detect many more spots in transfer of 2D-gels than silver staining (J Segers and M Rabaey, Protides of the Biological Fluids, 33:589, 1985). AuroDye™ Forte is intended to be used on nitrocellulose or PVDF membranes. Staining of proteins bound to nitrocellulose or PVDF membrane can be done by amido black, Ponceau or colloidal gold stain. In my experience, maximum sensitivity is achieved using colloidal gold stain. Proteins that are present in very minute amounts generally cannot be visualized by Ponceau or amido black stain.
AuroDye™ Forte Colloidal Gold reagent comes in a pack of 500 ml with 10 ml Tween-20, sufficient for about 15 standard sheets (10 x 15 cm, 30 ml/sheet). AuroDye™ Forte is ready for use and should not be diluted. It is to be stored at 2-8ºC. The other reagents required for the staining procedure are routinely used chemicals. Staining can be done even after performing immunodetection on membranes; the only requirement for this is to strip the membrane in destainer containing 40% methanol and 10% acetic acid for 30 minutes. The staining protocol is very quick. After transfer of proteins onto the nitrocellulose or PVDF, membranes are incubated for 30 min in PBS with 0.3% Tween-20 at 37ºC. After incubation, membranes are washed in PBS with 0.3% Tween-20 3 times for 5 min. Membranes are then incubated with AuroDye™ Forte colloidal gold reagent for 2-4 h, until optimum color formation is obtained. After incubation, membranes are washed with water to remove excess stain. After washing, membranes are air dried while the membranes are kept between sheets of filter paper. The stain does not fade. The colloidal gold can be reused 2 times depending on the required staining intensity and the quantity of protein on the membranes.
My research aims to identify proteins/tumor antigens which elicit an antibody response in cancer. It involves separation of tumor cell proteins on 2D gels followed by transfer of proteins from 2D gels to PVDF membranes. In order to detect proteins which elicit an antibody response in cancer patients, these PVDF membranes are immunostained with IgGs purified from sera of cancer patients, followed by chemiluminescent detection. The resulting film image shows reactive spots, which represent tumor antigens. To find out which spot on the film corresponds to which spot on the silver stained 2D gel, the film must be overlaid onto the silver stained 2D gel, but there are problems in overlapping images due to differences in the sizes of 2D gels. These differences occur because of the different conditions used during silver staining and the transfer of proteins from 2D gels to PVDF membranes.
This problem was solved with GE Healthcare’s AuroDye™ Forte Colloidal Gold reagent. Because 2D blots can be stained with colloidal gold even after immunostaining, film and colloidal gold stained 2D blot images can be overlapped. It is then easy to correlate spots from colloidal gold stained 2D blots to the silver stained 2D gels because colloidal gold stains all of the proteins on the 2D blots which can be visualized by silver staining. However, the appearance of some spots on colloidal gold stained 2D blots is different than on the silver stained 2D gels. This difference is a result of the different chemistries involved in the different staining methods. These differences are not great enough to create problems in comparing colloidal gold stained 2D blots with silver stained 2D gels.
AuroDye™ Forte Colloidal Gold reagent can also be beneficial for staining blots obtained by 1-dimensional electrophoresis, where small amounts of protein are typically loaded. The band intensity obtained by AuroDye™ Forte colloidal gold staining can also be used for quantification.
I would, therefore, recommend AuroDye™ Forte Colloidal Gold reagent for quick and sensitive detection of proteins on membranes, in spite of the marginally extra cost of colloidal gold.
Sanjeev Shukla
PhD Student
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Biochemistry and Cell Biology Department