Activation Assays

Assay Factor

Assay Considerations

Recommendations

Assay principle

Traditional Pull-Down

• Utilizes affinity beads coated with an effector protein to selectively bind the active GTPase.
• The signal is analyzed by western blot. 

Traditional pull-down assays use methods familiar to most labs and therefore provide a familiar point of entry.

The G-LISA assay is simple to use and has many advantages that should be considered (see below for comparisons).

G-LISA

• Uses a 96 well plate coated with an effector protein to selectively bind the active GTPase.
• The signal is analyzed by enzyme-linked immunosorbent assay (ELISA).

Equipment

Traditional Pull-Down

• SDS-PAGE apparatus
• Western blot apparatus

Consider the equipment requirements prior to deciding upon an assay format.

G-LISA

• Orbital plate shakers (2)
• Spectrophotometer (set to 490 nm)
• Multichannel pipette (recommended if handling high number of sample)

Starting material required per assay

Traditional Pull-Down

300 - 2000 µg per assay

If starting material is limited, e.g. primary cells, the G-LISA assay is required.

G-LISA

5 – 50 µg per assay

Cost

Traditional Pull-Down

Available as:

• Combo kit (10 assays each RhoA/Rac1/Cdc42)
• Starter kit (20 assays)
• Regular kit (50-80 assays depending on target protein)

Cost issues to consider:

Cost per assay: The G-LISA is the best value per assay.

Cost per kit: The traditional pull-down assay starter kits generally provide the lowest cost per kit.  NOTE: Consider the combo kit as a cost effective choice.

G-LISA

Available in 96 well format. Each well can be broken off and used individually.

Sample handling

Traditional Pull-Down

Centrifugation steps and western blot analysis makes this assay most suitable for low sample numbers (≤10).

<10 samples: either assay

>10 samples: G-LISA recommended

NOTE: Dose responses and time courses are often recommended and will greatly increase the number of samples being analyzed.

G-LISA

Amenable to high sample numbers (>10) or dose response/time-course analyses. Wells can be detached and used individually.

Quantification of results

Traditional Pull-Down

Western blot quantification has a very narrow linear range.  Also, multiple manipulations of the samples result in a somewhat variable assay.

G-LISA assays are recommended for simple, reproducible quantification of results.

G-LISA

Provides a simple numeric readout

Reagents required that are not in the kits

Traditional Pull-Down

• Cell or tissue lysate
• Protein quantitation reagent
• Anti-mouse secondary antibody
• SDS-PAGE buffers
• SDS-PAGE gels
• Molecular weight markers
• Western blot buffers
• Western blot detection reagents

The G-LISA assay provides all reagents necessary to perform 96 activation assays. Wells can be detached and used individually.

G-LISA

• Cell or tissue lysate