GTPases, pull-down assays and antibodies

The term GTPase, GTP-binding protein – or simply G-protein – is used for enzymes that are involved in cellular signal transduction by utilizing the nucleotide GTP.

They consist of 2 major subgroups called Heterotrimeric G-proteins and Small GTPases, and their activities are modulated by GEFs and GAPs in a cycling fashion (Fig. 1).

gtp_sigtrans_small

Figure 1: Find out more by clicking on the icons

Traditionally, measuring the activation state of a small GTPase in vivo (i.e. the GTPase-GDP/GTP levels) required tedious 32P-labelling of cells, enzyme isolation and HPLC of bound GDP and GTP. In contrast, non-radioactive pull down assays (Fig. 2) allow very easy determination, especially since GTPase Activation Kits for

  • Ras
  • Rap
  • Ral
  • Rac

are availability. All kits contain GTPase, their respective binding domain and the nucleotide cofactors GDP and GTPγS. After adding the binding domain to the cell extract the activated protein is pulled out and subsequently detected by Western blotting. This approach has greatly accelerated and simplified semi-quantitative activity determinations.

gtpase_activation_kits Figure 2: Pull down assay that exploits the selective interaction of Ras-binding domain (RBD) of the Ras-effector Raf with the active Ras-GTP conformation. GST-tagged RBD is added to extract Ras-GTP.

In addition to the kits that include all components required for GTPase detection and analysis in one box, single components are separately available. We offer specific monoclonal and polyclonal GDP/GTP Binding Antibodies of the Ras- and Rho/Rac/Cdc42 family. All antibodies are recommended for Western Blot, Immunohistochemistry, Immunofluoerescence, Immunoprecipitation (IP) and ELISA.

  • anti-Pan Ras
  • anti-Rap1
  • anti-RalA
  • anti-Rac1

GTP Signal Transduction – Heterotrimeric G-Proteins

Heterotrimeric G-Proteins are GTPases composed of α-, β-, and γ-subunits. These GTPases are classically associated with plasma membrane receptors containing seven transmembrane domains (G protein-coupled receptors [GPCRs]). Receptor activation activates the G-Protein by inducing the exchange of GTP for GDP at a binding site on Gα. Gα and/or Gβγ, then goes on to interact with effector proteins.

In mammals, about 20 Gα subunits (39 to 46 kD), about 5 distinct Gβ (ca. 35 kD) and at least 12 Gγ subunits (from 7 to 10 kD) have been identified.

Product Accession No. Cat. No. Amount  Downloads
GsαL
stimulatory heterotrimeric G-protein, long splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-501 1 ml
GsαL-Asn295
stimulatory heterotrimeric G-protein, long splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-502 1 ml
GsαL-Leu227
stimulatory heterotrimeric G-protein, long splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-504 1 ml
GsαL-Leu227-Asn295
stimulatory heterotrimeric G-protein, long splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-503 1 ml
GsαS
stimulatory heterotrimeric G-protein, short splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-505 1 ml
GsαS-Asn280
stimulatory heterotrimeric G-protein, short splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-507 1 ml
GsαS-Leu212
stimulatory heterotrimeric G-protein, short splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-508 1 ml
GsαS-Leu212-Asn280
stimulatory heterotrimeric G-protein, short splice variant of the α-subunit
rat, recombinant, Sf9 insect cells
NM_019132 PR-506 1 ml
Gustducin + β1γ2
Gustducin + G-protein β1γ2-subunits
rat, recombinant, Sf9 insect cells
X65747 PR-601 1 ml

GDP/GTP Binding Protein Kits

The GTPase Activation Kits provide rapid, cost-effective and reliable tools for the detection and semi-quantitative analysis of the cellular activation state of several GTPases.

Active GTPase (i.e. complexed to GTP), is purified from cell extracts via a one-step batch affinity purification step exploiting its affinity towards effector proteins that selectively interact with the active, GTP-bound GTPase conformation, which is consequently detected by Western blotting applying the included antibody.

The kits also include the appropriate recombinant GTPase proteins that, once preloaded with GDP or GTPγS (or GppNHp, respectively), can be used to “spike” cell extracts, thus serving as an internal control of signal specificity.

Product Cat. No. Amount Downloads
Ras Activation Kit
Detection of cellular Ras-GTP
Ras GTPases
PR-950 10x 6 assay points
Rap Activation Kit
Detection of cellular Rap-GTP
Rap GTPases
PR-951 10x 6 assay points
Ral Activation Kit
Detection of cellular Ral-GTP
Ral GTPases
PR-959 10x 6 assay points
Rac Activation Kit
Detection of cellular Rac-GTP
Rac/Rho GTPases
PR-962 10x 6 assay points

GDP/GTP Binding Protein Antibodies

Guanosine diphosphate (GDP) / guanosine triphosphate (GTP) binding protein activity is regulated by the binding and hydrolysis of GTP. These proteins are crucial for signal transduction events resulting in cell division, cytoskeleton management, and sensory perception. We offer very specific monoclonal and polyclonal affinity purified antibodies out of the Ras- and Rho/Rac/Cdc42 Family for Western Blot, Immunohistochemistry, Immunofluoerescence, Immunoprecipitation (IP), and ELISA.
Product Cat. No. Amount Downloads
anti-Pan Ras
mouse, monoclonal antibody
ABD-047 50 µl
anti-Rap 1
rabbit, polyclonal antibody
ABD-046 50 µl
anti-Ral A
mouse, monoclonal antibody
ABD-048 50 µl
anti-Rac 1
rabbit, polyclonal antibody
ABD-056 50 µl

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