Would you mind suggest me some techniques to maintain my fused proteins` stability and function? My proteins are fused fluorophore proteins loosing their fluoresence substantialy at +4c.
Also, is there a modified EMSA to detect protein-protein interaction?
If the proteins are unstable in the storage conditions at 4oC, try storing them frozen at -20oC or -80oC (using flash-feezing technique), or storing them unfrozen at -20oC in 50% glycerol. Make sure they are not exposed to light during storage, which could cause bleaching. Check to see if they are becoming degraded by proteolysis by running SDS-PAGE. If so, they need further purification to remove proteases, and make sure to include protease inhibitor cocktail during purification.
What fluorescent tag is on the protein? Also what is the conjugation chemistry? It may be possible that the fluorophore is bleaching (in which case you want to protect it from light). There may be aggregation and quenching of the fluorophore. You can test this by running the degraded product on a gel. Is the untagged version of this protein stable under the same conditions?
Dear Safaa,
What about to optimize the gene you are expressing. For the expression in insect cells we order synthetic genes optimised for proper expression, mRNA stability and so on.
If the proteins are unstable in the storage conditions at 4oC, try storing them frozen at -20oC or -80oC (using flash-feezing technique), or storing them unfrozen at -20oC in 50% glycerol. Make sure they are not exposed to light during storage, which could cause bleaching. Check to see if they are becoming degraded by proteolysis by running SDS-PAGE. If so, they need further purification to remove proteases, and make sure to include protease inhibitor cocktail during purification.
Thank you so much for y`all!
My proteins are eGFP-Nrf2 and Keap1-mcherry. Untag protein is the same, not stable at +4c. I want to store the proteins and maintain their activities. Does store them frozen affect their activity.
Safaa, you got some troubleshooting to do here. If the untagged protein is unstable too, please try to purify them to remove proteases. Once they are gone, the proteins should be stable (assuming it is the proteases). With fluorescent protein fusions you always want to be careful with freezing below 4C since they aggregate if not stored in appropriate manner (flash freezing, glycerol etc.). To test this you can run the stored proteins on a PAGE gel and see what condition is giving you the desired band corresponding to the un-aggregated protein both in the tagged and untagged conditions. Hope it works out! Good luck troubleshooting!
Just a shot in the dark, but many proteins are stable as ammonium sulfate precipitates. These shouldn't freeze solid at -20C and if you can dilute them out far enough or buffer exchange before hand it may work for your applications.
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