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Welcome.
A few things - first check that your plasmid is OK - run some on a gel and make sure that it is largely intact and not degraded.
Could the gene be a membrane associated protein? If so, you may need to change how you solubilize the cells when making lysates. Try lysing in 2x Laemmli buffer and boiling straight away.
How strong is the reducing buffer for your running?
Do you see any aggregation of proteins after boiling the lysate?
How big is the protein? (what gel% are you using?)
#142146 Cloning & Expression Problem in HEK-293Ts
Posted
on 24 September 2012 - 12:40 PM
