Add Reducing Agent to SDS-PAGE Sample Buffer for Western Blot

[sotellme]

should we add a reducing agent (DTT or 2-Mercaptoethanol) or not in the SDS_PAGE's sample buffer? The plan is using this gel for Western blot.

 

[quetzalcoatl9]

should we add a reducing agent (DTT or 2-Mercaptoethanol) or not in the SDS_PAGE's sample buffer? The plan is using this gel for Western blot.

most ppl use mercaptoethanol, and yes you probably should for what you want to do.

the point of the reducing agent is to break any disulfide bonds that may exist in your protein. if you don't your gel will smear. proteins running on an SDS-PAGE should be as close to linear as possible so that they migrate through the PAGE matrix appropriately.

also, the cell is typically a reducing environment so reducing agents like dtt or bme are common in keeping proteins in their native state (or preventing aggregation) until use. any cystines exposed to the cytosol will probably be reduced - the addition of sds detergent should then "open up" the protein so that every cysteine is reduced (this is not the main reason for the sds, but is important nonetheless).

but also be sure to check that your protein is not made of subunits - if it is then they will run on the gel seperately, and you will probably wind up running them off.

 

[ravenprp]

It is recommended to add a reducing agent, such as DTT or 2-Mercaptoethanol, to the SDS-PAGE sample buffer for Western blot experiments. This is because reducing agents help to break disulfide bonds, which can interfere with protein separation and detection on the gel. By adding a reducing agent, the proteins will be denatured and have a uniform negative charge, allowing for more accurate separation based on size during electrophoresis. Additionally, reducing agents can help to prevent protein aggregation and degradation, ensuring that the protein of interest is intact and detectable on the Western blot. Overall, the addition of a reducing agent to the SDS-PAGE sample buffer is crucial for obtaining reliable and reproducible results in Western blot experiments.