The traditionally-used Coomassie Brilliant Blue stain is sufficient for routine protein gels. However, the detection limit of this stain is 0.1 ug. When sensitivity becomes an issue, one needs to switch to silver staining, which is cheaper than fluorescent stains and can detect protein concentrations that hit the nanogram levels. Preparation of silver staining solutions is a cumbersome process. Solutions should be prepared fresh every time and they must be prepared carefully so that reproducible results with a clear background are obtained. For these reasons and in order to avoid wasting precious protein samples in gels that do not stain well, I find using the GE Healthcare’s (formerly Amersham Biosciences) PlusOne™ Silver Staining Kit rather advantageous and cost-effective.
This kit is very sensitive and convenient to use. The procedure is fast, easy and highly reproducible. This kit also offers several fold more sensitivity than routine Coomassie staining. The kit contains almost all of the chemicals required for staining. However, some basic laboratory chemicals (acetic acid, trichloro acetic acid, glycerol etc.) are not provided in the kit. The protocol supplied along with the kit provides a step-by-step staining guide for protein gels. Freshly prepared solutions have to be used in order to avoid a dark background. Water used for the staining should be of high quality and gloves should be worn while handling the gels.
The kit can be used only for staining denaturing/non-denaturing protein gels and cannot be employed for DNA gels, for which a separate kit is available from the manufacturer. Gels containing urea can also be stained using this kit. It can be used for both pre-cast gels and conventional slab gels. I have used this kit for staining mainly 2D gels, wherein sensitivity becomes an issue. Using this kit, I find the gels to be uniformly stained without dark patches. I have obtained a transparent background for all the gels I have stained with this kit. I have also used this kit to check protein purity and integrity (as judged by counting the number of bands and performing the densitometric analysis to measure the signal/noise ratio) whenever I prepare a fresh protein stock for crystallization.
The contents of the kit are sufficient for staining 10 pre-cast gels of 12.5 x 26 cm or 20 mini slab gels of 10 x 10 cm. Different protocols are used for staining different kinds of gels and therefore, the time required to stain gels can range from 2 to 8 hours. This time includes fixation, washing, sensitization, the silver reaction as well as development and stopping the reaction. The silver stained spots are not compatible with mass spectrometry because of the use of glutaraldehyde and formaldehyde in the solutions. Use of these reagents can be omitted if a mass spectrometric analysis of the spots is required. However, this compromises the sensitivity of the staining reaction.
The shelf life of the kit is approximately one year and it should be stored at room temperature. All steps are performed at room temperature. Racks for staining gels are also available from the manufacturer. However, only glass trays should be used otherwise.
Vikas Jain
Research Scholar
Indian Institute of Science
Molecular Biophysics Unit