Hi everyone, during sample prep, we usually boil the samples with 2-mercaptoethanol in Laemli buffer. However by mistake, some of the samples were boiled with Laemli that had SDS but no 2-mercaptoethanol. Then I found out and add the 2-mercaptoethanol to the samples immediately after they cool down. Can we still detect the proteins with the antibodies like usual?
Hi,
heat up the samples to 95 °C, cool them to room temperature, and check for precipitate. If there is no precipitation, samples could be good.
The addition of 2-mercaptoethanol is important because it prevents the creation of disulfide bonds and thus prevents protein aggregation. SDS keeps proteins in their primary structure but does not prevent the creation of disulfide bonds between peptide chains.
Since 2-mercaptoethanol/DTT work at RT as well thus you can add them after the samples have been boiled and cooled as well.
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