Syk Western Blot

Using a Western blot, we will evaluate at what level our lab stock of HEK293 cells express Syk relative to the amount of Syk we plan to introduce as part of our BCR Tango system.

Knowing whether or not our HEK293 cells express Syk will better enable us to determine what level of Syk expression we need in our HEK293 model system and how much cross-talk we can expect from recruitment of endogenous Syk to the BCR upon antigen binding.

pEXPR TRE: Syk-TEV or pEXPR TRE: Syk-eYFP

anti-Syk antibodies + secondaries

anti-GADPH antibodies + secondaries

Cell populations:

Is including cells without pEXPR TRE: Syk-TEVp a useful control? If so, do we need a dox ladder for those cell populations?

Since Syk-TEVp should run at a different band size than endogenous Syk, we should be able to identify the presence of both or either on a Western blot using anti-Syk antibodies. (There should be two bands, one corresponding to Syk-TEVp and the other to endogenous Syk.) We can use GAPDH (which we assume is produced at the same level in all of the cell populations we are testing) to normalize our data. This way, we can make comparisons between different cell populations while controlling for the amount of protein that we loaded in each lane. After washing the paper blot with anti-Syk and anti-GADPH, we should get three bands based on the sizes of the proteins:

From the intensity of these bands, we should be able to determine the relative levels of endogenous and exogenous Syk expression.