I am performing a 2D-DIGE experiment using 50 ug of protein to each samples. In total, I have 150 ug of protein per gel. I would like to use "blue silver" stain and pick up the spots from gels manually. Then, I will to use a MALDI-TOF/TOF to identification.
I believe that using "blue silver" and only 150 ug of proteins per gel is not enough to detect the low abundance proteins. What is better? Run non-labelled proteins in the same gel with labeled proteins (Cy2/3/5) or run in separated gels.
Sorry to say that 2D-PAGE is not a sensitive method to detect low abundant proteins. You can try some short-gun proteomics (LC-MS/MS). If you use in vitro model, SILAC is one of the best sensitive methods. For in vivo, spectrul LC-MS/MS a good option.
If you load higher amount of proteins in 2D PAGE, it will hinder protein separation. Don't use too much. Even you can use other types of stain. Before that be sure about stain-protein interactions. Some interaction can damage proteins and cannot be detected by MALDI-TOF.
World is directing toward more to shotgun method rather than gel base proteomics. Check the following article, specially protein analysis section for details:
http://www.sciencedirect.com/science/article/pii/S1359644613003954
Dear Glaucio,
if you use 2D-DIGE, I suppose that you want to monitor differences between samples from two different conditions. Logically you mixed 50 µg from each sample (2 x 50 µg), each one labelled with a specific dye + the mix of 25 µg from each as an internal control. The spots of interest are then picked up and studied by MS/MS. If you did it this way, why do you want to "blue silver stain" the gels afterwards ? I do not believe that you will have better information about differences, compared to the dyes.
Thank by the answers.
Dear Saifur,
I understand that 2D-DIGE is less sensitive compared to shotgun proteomics. However, unfortunately, I do not have the equipaments required to carry out this approach.
Dear Yannis,
I will perform all my analysis using the fluorescence signal obtained with the Cy2/3/5 dyes. However, I need to use a post stain, as blue silver, to visualise the spots that I will pick up manually.
OK, now I understand the problem if you cannot use a fluorescent scanner to pick up the spots of interest.
Hi,
in our lab we normally run a prep gel with at least 500 µg of protein after the analytical gels are done. We use the G-Dye 100 to label 50 µg to load on the gel and do not stain after that. In our lab time is an important factor so we decided to skip staining completely and pick with the blue fluorescent image -> the picking guide you will find here http://www.dyeagnostics.com/site/en/products/refraction-2d/.
Kind regards,
Kristin
Please read the following articles and review:
El-Ashram S, Yin Q, Liu H, Al Nasr I, Liu X, Suo X, et al. (2015) From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph). PLoS ONE 10(12): e0143232. doi:10.1371/journal.pone.0143232
El-Ashram S, Sun X, Yin Q, Liu X, Suo X (2015) Exploring Early and Late Toxoplasma gondii Strain RH Infection by Two-Dimensional Immunoblots of Chicken Immunoglobulin G and M Profiles. PLoS ONE 10(3): e0121647. doi:10.1371/journal.pone.0121647
S. El-Ashram, Q. Yin, J.R. Barta, J. Khan, X. Liu, and X. Suo, Immunoproteomic technology offers an extraordinary diagnostic approach for Toxoplasma gondii infection. Journal of Microbiological Methods 119 (2015) 18-30.
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