assays

[zeny]

what are the pros and cons of using an enzyme assay kit over immunoblot experiments?

[CharonY]

Well, direct comparisons are not easy as there are a host of different enzyme assays, each with different ad- and disadvantages.

In general terms immuniblots can be adapted to all kind of proteins, provided you can get a good antibody, whereas enzyme assays require that the protein under investigation has some quantifiable enzyme activity.

[Tr0x]

Immunoblots are generally the first step to confirm that your protein is physically "there" (i.e. in your sample/being produced by your system)

Pros:

• Quick and relatively cheap (dependent on how expensive your Abs are)
• Quantifiable if compared against known concentrations of your protein of interest

Cons:

• Doesn't give you any indication of activity, only showing the total amount of protein present
• Heavily dependent on the accuracy of your Abs

Enzyme assay kits are useful if your protein of interest is known/relatively common/a substrate is known.

Pros:

• Usually quick, and very reliable results on the activity of your protein product

Cons:

• Taylor-made to suit your protein product, meaning it has to have known activity (you can of course devise your own assay if you know a substrate)
• Can be more expensive, and usually requires additional equipment (i.e. plate reader)
• Only shows how much of the protein is active, and not the total amount produced

These assays are generally complementary, and it's rare to see a paper without some form of both.