LaCava Research Wiki

If you want to know more about protein staining:

• Gauci, V. J., Wright, E. P. & Coorssen, J. R. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J Chem Biol 4, 3–29 (2011).
• Harris, L. R., Churchward, M. A., Butt, R. H. & Coorssen, J. R. Assessing detection methods for gel-based proteomic analyses. J Proteome Res 6, 1418–1425 (2007).
• Miller, I., Crawford, J. & Gianazza, E. Protein stains for proteomic applications: Which, when, why? Proteomics 6, 5385–5408 (2006).

and someone's protocol https://doi.org/10.1016/j.pep.2014.12.006

CBB IR fluorescence can be also used for a better sensitivity. We haven't made a protocol yet, but we want to test diffirent excitation and emission wavelenths: excitation - 600nm (470 and 685 didn't work well) emission - 720nm (LP filter should be ok)

ImageQuant TL - analysys is pretty straightforward, there is a good manual (link) and a youtube tutorial (https://youtu.be/sQmDEwo-R4M) , but I made a little guide:

1. Run ImageQuant TL
2. Open your gel scan (*.tiff/*.gel)
3. Choose stepwise analysis
4. Create lines (automatic mode works pretty well; you can change the shape of your lines on the next step if you are not satisfied with the result)
5. Adjust (move and bend) strating and finishing lines of your gel if: starting and finishing lines are not in the gel; some parts of the gel front migrated at different speeds; there are chips/holes/spots of bromphenol blue at the edge of the gel (background subtraction is sensitive to this kind of defects)
6. Adjust the witdh of your lanes
7. Do the background subtraction: use rolling ball (diameter ~85 usually works fine) if your bands have the same witdh. Rubber band is less sensitive to the difference in bands' width, but is much worse at subtracting the background.
8. Automatic band detection doesn't work well, so my advice is choose the peaks of interest manually; you can left click both on the gel scan and the gel profile (right click on a band to remove it). Consider using fixed peak width when you compare the same band on different lines.
9. Do the MW calibration: choose the existing set of markers
10. or create a new one by clicking on "Edit"
11. Click "Edit/create Standards" and add the MW used in your marker mix
12. Left Click on the line you want to use as a marker
13. You can choose more than one marker line if you see that some lines migrated in a different way (you can also remove the marker line with a right click; sometimes adding a new line makes the calibtration worse)
14. Use Log Curve for calibration and click compute
15. Do the quantity calibration: right click on the bands with known amount of protein (in this case - 100, 250, 500ng) and click on Calibrate. I recommend to use four standards for in your expected range of concentration (for example, take 25, 50, 75, 100ng for the gels with 15-150ng of protein)
16. In some concentration ranges BSA standards should fit to linear curve almost perfectly
17. But in some cases you can also try using other functions (quadratic, for instance)
18. Forcing through origin helps you estimate concentration way below your last standard
19. Skip the normalisation step
20. After you have completed all the steps you can jump to any step if you want to add something to your analysis
21. N: you can add some bands
22. Export all your data as a .txt file: choose the tab "Comparison", then "Edit" - "Export to file"
23. Save as .txt
24. Copy and paste the data to excel table. Rows and columns will be properly aligned