Detecting proteins in polyacrylamide gels and on electroblot membrane

1. Detecting proteins in polyacrylamide gels and on electroblot membrane 2001-20487 Kim Sang Hoon

2. Contents • Introduction • Organic dyes and silver stains • Reverse stains • Colloidal dispersion stains • Organic fluorophore stains • Metal chelate stains

3. Introduction • History of stains used to visualize proteins • Bromophenol blue Amido black Coomassie blue R-250 Coomassie blue G-250 Silver staining Colloidal staining Negative staining SYPRO Red, Orange, Ruby staining

4. Organic dyes • Organic dyes • Coomassie blue: wide spread popularity since its introduction in the early 1960s, capable of detecting as little as 30-100ng • Amido black : largely relegated to medium sensitivity, colorimetric detection of electro blotted proteins on PVDF and nitrocellulose membranes

5. Coomassie blue

6. silver stains • Silver staining • Detection sensitivity: 0.6-1.2ng • Alkaline/silver diamine and acidic/silver nitrate are most commonly used • Alkaline/silver diamine: histological producers and use ammonium hydroxide to form soluble silver-diamine complex • Acidic/silver nitrate: photographic procedures and rely upon gel impregnation with silver ions at acidic pH

7. Silver staining

8. Reverse stains • Reverse stains were developed specifically to improve protein recovery from polyacrylamide gels • Staining is quite rapid, usually requiring 5-15min to complete and the biological activity of proteins is often preserved

9. Zinc staining

10. Copper staining

11. Colloidal dispersion stains • The stains contain fine particles with high affinity for protein • The stains are not generally used for detection of proteins in polyacryl- amide gels • India ink stain: simple to perform and inexpensive • Colloidal metal stains: gold staining

12. Organic fluorophore stains • Fluorescent detection of proteins after electrophoresis is gaining popularity with laboratories engaging in large-scale proteome research • Covalently bound fluorophores: more recently propyl-Cy3 and methyl-Cy5 dyes have been utilized • Noncovalently bound fluorophores: SYPRO Orange and SYPRO Red

13. SYPRO Orange staining

14. SYPRO Red staining

15. Metal chelate stainig • The metal chelate are a relatively new family of protein visualization reagents developed to be compatible with modern proteomics research • Colorimetric metal chelate stains: disulfonate/ferrous(600ng), copper phthalocyanine tetrasulfonate(10-20ng) • Luminescent metal chelate stains: SYPRO Ruby 2-DE and IEF stains allow one-step, low background staining of proteins in polyacrylamide gels without resorting to lengthy destaing steps.

16. SYPRO Ruby staining

17. Porperties of an ideal general protein stain • The seven Ss of superior staining • Safety • Sensitivity • Simplicity • Specificity • Speed • Stability • Synergy

18. Table (bio-rad)

19. 2-D Staining

20. Conclusion • With the development of proteomics into a high-throughput approach for the study of global protein regulation, new demans are being placed on protein visualization methods • Newer stains such as Zinc-imidazole, and the noncovalently fluorescent stains (SYPRO dyes)are available to meet the growing demand of mordern protein microcharacterization technologies