Can I use Progenesis SameSpots for analysis of secondary stained gels?
Yes. Secondary staining, including western blotting, of a 2D gel gives you the power to characterise proteins showing statistically significant differences. This kind of validation can be easily added to your 2D gel analysis results using Progenesis SameSpots.
Application Note – Compare Western Blots and
2D gels to characterise proteins
Comparing two images that are very different in terms of spot patterns and the intensity of the same spot measured under different staining conditions is a big challenge. Progenesis SameSpots applies highly accurate pixel-level alignment so you can directly compare 2D gels with secondary stained images including:
- Phosphotproteins e.g. Pro-Q® Diamond
- Glycoproteins e.g. Pro-Q® Emerald
Why do secondary staining of your 2D gels?
Secondary stains can identify a protein of interest within a complex mixture and confirm the size, expression level and presence of post-translational modifications. Antibodies also provide >10-100-fold sensitivity compared to total-protein stains1, allowing them to detect very low abundance proteins. This means secondary staining has applications in discovery and development phase research as well as bio-processing:
- Characterise discoveries and validate your hypothesis in proteomics experiments2-4
- Check specificity of antibodies for therapeutic or commercial use5
- Quality control the production of biologics and recombinant proteins5
Many customers are using Progenesis SameSpots to discover proteins that are differentially expressed. Identifying these discoveries and validating a result using alternative techniques is often carried out as part of the same experiment. This validation can be a condition of further research funding or publishing data.
Benefits of using Progenesis SameSpots
- Flexibility to set up different image groups for qualitative and quantitative comparisons within the same experiment
- Same analysis process applied to all images, making it easy to use and incorporate into your study
- Compare 2D gel spot patterns with secondary stained images directly
- Visualise images compared in the same view and export figures for publication
Progenesis SameSpots detects the presence and location of a His-tagged protein within an E. Coli cell free extract on a 2D gel (top) and blotted onto a nitrocellulose membrane (bottom).
Highly accurate pixel-level alignment means you can directly compare differentially stained images. The western blot image (green) has been aligned to the 2D gel image (red).
Set up different image groups for qualitative and quantitative comparisons within the same experiment.
Supporting information available to demonstrate how to perform analysis based on a "multiplex without using internal standard" experiment set-up.
Application Note – Comparing Western Blot and 2D gel images to Characterise Proteins
Software “How To” – Creating a new Multi-Stain experiment
- Kendrick N, Johansen J, Hoelter M.2D gel Phosphoprotein Western Blotting. PDF available on-line from Kendrick Labs Inc http://www.kendricklabs.com/PP_PhosphoProteinWB.pdf
- Zhao Z, Liu J, Shi B, He S, Yao X, Willcox MD. Advanced glycation end product (AGE) modified proteins in tears of diabetic patients. Mol Vis. 2010 Aug 11; 16:1576-84.
- Starosta V, Pazdrak K, Boldogh I, Svider T, Kurosky A. Lipoxin A4 counterregulates GM-CSF signaling in eosinophilic granulocytes. J. Immunol. 2008 Dec 15; 181(12):8688-99.
- Dhiman M, Nakayasu ES, Madaiah YH, Reynolds BK, Wen JJ, Almeida IC, Garg NJ. Enhanced nitrosative stress during Trypanosoma cruzi infection causes nitrotyrosine modification of host proteins: implications in Chagas' disease. Am. J. Pathol. 2008 Sep;173(3):728-40.
- Johansen J, Hoelter M, Kendrick N. Optimization of 2D Gel Transblotting for Host Cell Protein Analysis. PDF available on-line from Kendrick Labs Inc http://www.kendricklabs.com/PP_NK_AES-09Biologics.pdf
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