Hello everyone, I was planning to run a FRET experiment to characterize in more detail how my protein of interest inserts the membrane (based on a simple membrane mimicing vesicle model). I was advised to use the two following dyes: Cy5 and ATTO448. I performed the labelling procedure but have a huge problem removing the excess of the dye, especially Cy5. I tried to use the SulfoLink Resin Coupling (Thermo Fisher) columns, but the result was poor and it was not possible to run the experiment. Then, instead of using the columns I run the 24h dialysis followed by the SEC (Superdex 75). The protein in its monomeric state is about 5kD and the dye's MW is approximately 1kD so I expected to separate protein from the free dye even though the recommended separation range for the Superdex 75 is > 3kD. The purity of samples improved but unfortunately, the free dye was still present at relatively high comcentrations in almost all collected fractions. Has anybody had similar difficulties removing the excess of free Cy5 and ATTO488 from samples? Any suggestions?
You might get better separation with Superdex 30, which has a lower molecular weight fractionation range than Superdex 75. You should also consider hydrophobic interaction chromatography and ion exchange chromatography. These would probably be more effective than gel filtration.
Hi Sylwia,
Here are few possible solutions for you and I am mentioning them in an order I perform during the reaction.
1. Try to avoid high concentration of the dye. Dye concentration 3 to 5 times of the protein is good enough for labeling reaction.
2. After the reaction add 10X (to protein) BME in the reaction mixture. BME neutralizes the excess dye in the medium and mekes removal easier.
3. Use sephadex G25 or similar column (find the appropriate column depending on the protein mol. Wt.) to remove some excess dyes. If you run the G25 after labelling you can clearly see a faint band eluting first followed by a dense colored band. The faint colored band is the labelled protein. It works good for my ~10 kDa proteins. Bigger the protein better will be the result. you can run a test with the protein only to see upto which volume the protein elutes. Follow same amount of collection volumes after labeling.
4. Then use the G25 eluents for further purification either by mono_Q or mono_S columns (choose depending on protein charges at working pH). These columns are excellent but very costly. If you don't have these use RP-HPLC to remove excess dye.
RP-HPLC also works excellently for these dye separation processes. Another advantage of doing RP-HPLC is the eluent can be directly taken to the mass spec characterization as the fraction will have no salts.
These steps worked for me in many labelling reactions. Hope this will work for you as well. Let me know how successful these steps are.
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