How should I store small-GTPase pull-down affinity beads?

Lyophilized beads are shipped at room temperature. Upon arrival, beads can be stored desiccated at 4°C. After rehydration the beads should be aliquoted into experiment sized amounts and stored frozen at -70°C. Repeated freeze/thaws are not recommended.

What is the typical amount of lysate needed for activation assays?

For G-LISA-10-50 µg, for pull-downs-300-1000 µg. Making the G-LISA an enabling assay where lysate is limiting such as in primary cell cultures.

What are the major families of small G-proteins?

Small G-proteins are divided into five main families based on sequence and function: Ras controls cell growth and differentiation; Rho regulates actin cytoskeleton and cell motility; Rab directs vesicle trafficking; Ran manages nucleocytoplasmic transport and mitosis; and Arf facilitates vesicle budding and lipid metabolism.

How do I load a small GTPase protein with nucleotide?

To load a small GTPase with GDP (inactive) or non-hydrolysable GTPγS (active), EDTA is used to chelate magnesium, causing nucleotide release. Excess GDP or GTPγS then binds the GTPase. Finally, magnesium is reintroduced to lock the nucleotide in place, enabling use in pull-down assays as controls.

How do I optimize western blot protein transfer for small G-proteins?

Ras superfamily small G-proteins are approximately 20–25 kDa in size. Because of their small molecular weight, they can easily pass through the transfer membrane during western blotting. Therefore, efficient transfer to a PVDF membrane is one of the most critical steps for obtaining a strong signal during subsequent antibody detection. The following steps are recommended: 1- Run & equilibrate the gel • Avoid low percent gels, 12-15% range or a 4-20% gradient gel is recommended. • Equilibrate the protein gel for 15 minutes at room temperature in western blot transfer buffer containing 15% methanol: Transfer Buffer 25 mM Tris, pH 8.3 192 mM glycine 15% methanol 2- Transfer proteins to PVDF membrane • A 0.2 µm rather than a 0.45 µm membrane pore size is recommended. • Transfer the proteins to a PVDF membrane at 4°C for 45 minutes at 75 V. TIME & VOLTAGE are critical to efficient transfer. • Semi-dry transfer is not recommended. 3- Wash the membrane •Wash the membrane once with TBS at room temperature: TBS 10 mM Tris, pH 8.0 150 mM sodium chloride 4- Dry the membrane • Allow the membrane to dry for 20–30 minutes, or overnight, at room temperature. This step improves protein binding to the PVDF membrane. 5-Rehydrate the membrane • Rehydrate the PVDF membrane in 100% methanol for 2–3 minutes. 6- Equilibrate in TBST • Transfer the membrane to TBST for 5 minutes at room temperature to equilibrate: TBST 10 mM Tris, pH 8.0 150 mM sodium chloride 0.05% Tween-20 7- Proceed with immunodetection • Continue with the blocking and antibody incubation steps according to the kit instructions or standard laboratory protocol.

When using tissue culture based expeiments, how should I determine the optimal timing and concentrations for GTPase activators in G-LISA ® and pull-down assays?

GTPases are generally activated very rapidly and transiently upon stimulation, with maximal activation ranging from 30 seconds to 30 minutes before declining to basal levels. Using a single time point, particularly when starting a project, you are likely to miss the activation peak. Recommended time points for a first experiment are 0, 1.5, 3, 6, 10, and 30 minutes. Additionally, a concentration-response curve for activators or inhibitors should be considered. Timepoint samples can be aliquoted and snap frozen for later analysis. Frozen lysates should be stable for several months at -70°C.

RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays

Product Description

Applications

Combo kit for detection of activated (GTP-bound) Cdc42/Rac1 and RhoA protein in cell and tissue lysates
Compare Cdc42, Rac1 and RhoA signaling pathways

Material

Kit contents (3 x 10 assays)

Rhotekin-PBD beads for affinity purification of active RhoA, B & C
RhoA-specific antibody to detect activated RhoA only
His-RhoA control protein
P21 activated kinase1 protein (PAK)-PBD beads for affinity purification of active Cdc42 & Rac
Cdc42-specific antibody to detect activated Cdc42 only
His-Cdc42 control protein
Rac1-specific antibody to detect activated Rac1 only
His-Rac1 control protein
Cell lysis buffer-optimized for maintenance of GTP-bound GTPases
Wash buffer-optimized for maintenance of GTP-bound GTPases
Loading buffer: for control reactions
STOP buffer: for control reactions
GTPγS: for control reactions
GDP: for control reactions
Protease inhibitor cocktail
DMSO: for protease resuspension

Equipment & materials required

Cell lysates
Cell scraper for harvesting cells
Liquid nitrogen for snap freezing lysates
Equipment for Western blot performance & analysis
Microcentrifuge

Biological Activity Assay

The BK030 kit is a combination sampler size of the Rac1, Cdc42 and RhoA pull-down kits. There is sufficient reagent to perform 10 pull-down assays for each GTPase.

Key characteristics

Semi-quantitative assay
Detection sensitivity: See BK035, BK034 & BK036 specifications
Detection specificity: Rac1, Cdc42 & RhoA proteins
Input requirements: 200 µg to 1mg of cell or tissue lysate per assay
Timeframe: Approximately 10 hours, typically performed over two days

Frequently Asked Questions

Kit contents (3 x 10 assays)

Rhotekin-PBD beads for affinity purification of active RhoA, B & C
RhoA-specific antibody to detect activated RhoA only
His-RhoA control protein
P21 activated kinase1 protein (PAK)-PBD beads for affinity purification of active Cdc42 & Rac
Cdc42-specific antibody to detect activated Cdc42 only
His-Cdc42 control protein
Rac1-specific antibody to detect activated Rac1 only
His-Rac1 control protein
Cell lysis buffer-optimized for maintenance of GTP-bound GTPases
Wash buffer-optimized for maintenance of GTP-bound GTPases
Loading buffer: for control reactions
STOP buffer: for control reactions
GTPγS: for control reactions
GDP: for control reactions
Protease inhibitor cocktail
DMSO: for protease resuspension

Equipment & materials required

Cell lysates
Cell scraper for harvesting cells
Liquid nitrogen for snap freezing lysates
Equipment for Western blot performance & analysis
Microcentrifuge

RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays