2DE map of rat testes were prepared and stained by silver nitrate. I also isolated the spots from the gels but one of my friends advised that "you must run a preparative 2DE and stain it by CBB to find better results".
What are your ideas?
2D gels can be stained by silver staining protocol. Once your gels are analysed and spots are picked, there is a protocol to destain the excised spot by using potassium ferricyanide and sodium thiosulphate for a certain time. This protocol differs from that of conventional commassie blue stained spots by kind of chemicals used for destaining. Once the gels are destained, they are washed several times using distilled water to remove the trace of the above chemicals used. Later on the spots were treated by trypsin for peptide digestion and were analysed by MALDI. I hope it will encourage you to proceed with the silver stained gels :)
In my point of you, the main problem remain the amount of materials used to perform 2D gel. I do silver staining 2D gel with 10 to 30µg of proteins. For CBB, I use between 100 to 500µg. The identification is easier using more materials.
A complex point is the correlation between "silver" 2D gel and CBB 2D gel. Sometime, it is complex to associate a "silver" spot to the "CBB" spot, as the staining and the sensitivity of each protein is different in both methods.
Besides the topics commented by Santhosh and Stéphane regarding the total amount of protein you may have on each protein spot stained by silver, usually the spots stained by silver may have higher level of contaminants due to more expose of the gel to contaminants (keratin, etc.) during the protocol of gel staining. To avoid this problem to interfeer with the protein identification I recommend you to do more washing steps during the first steps of the in gel digestion protocol. There are protocols available for the analysis of spots extracted from 2D gels stained with silver nitrate and usually they work fine when you use Mass spectrometers of high sensitivity, such as Orbitrap. I recommend the following protocol if you want to do in gel protein digestion, but again I recommend you to perform more washing steps.
Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Andrej Shevchenko,Matthias Wilm,Ole Vorm, and, and Matthias Mann*
Analytical Chemistry 1996 68 (5), 850-858.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics