Hello! I am not sure whether or not my protein samples are reduced or not. Based on the information below, can someone please let me know if they are reduced?
Sample setup:
__ uL total cell lysate (exact amount calculated from BCA assay)
1 uL Thermo Fischer 10X Sample Reducing Agent
2.5 uL Thermo Fischer 4X LDS Sample Buffer
1X RIPA buffer to dilute sample to 10 uL
Dear Kristen,
Protein samples are typically reduced before loading onto SDS-PAGE using a loading buffer containing a reducing agent such as DTT or b-mercaptoethanol (and the sample is also often boiled to further denature the proteins).
If you dilute your protein sample first with RIPA buffer and then add Sample Reducing Agent (and perhaps boil it for 60 - 90 seconds) all of the proteins should be reduced. You can then add some of the Sample buffer and load directly onto your SDS-PA gel for electrophoresis. (I am sure that Thermo Fisher would have supplied a standard protocol for this!).
You should take care to keep all of the buffers at the correct temperature, and always check that the Reducing Agent always smells a bit like rotting eggs as DTT and b-ME go off quickly (I always store these in the fridge but Thermo Fisher may recommend freezing the Reducing agent).
Andrew