Cdc42 Pulldown Activation Assay Kit

Hersteller Cytoskeleton
Kategorie
Typ Assay
Specific against other
Menge KIT 50 assays
ArtNr BK034
eClass 6.1 32161000
eClass 9.0 32161000
Lieferbar
Delivery Time
1-2 Weeks
Shipping Temp.
AT
Storage on Arrival
4C
Citations

Sakabe et al., 2012. Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells. Immun. Ageing. v 9, p 2.

Nithipatikom et al., 2012. Cannabinoid Receptor Type 1 (CB1) Activation Inhibits Small GTPase RhoA Activity and Regulates Motility of Prostate Carcinoma Cells. Endocrinology. v 153, pp 29-41.

Asai et al., 2011. Involvement of Rac GTPase activation in phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to ICAM-1. Biochem. Biophys. Res. Commun. v 406, pp 273-277.

Abu-Elneel et al., 2008. A delta-Catenin Signaling Pathway Leading to dendritic protrusions. J. Biol. Chem. v 283, pp 32781-32791.

Nur-E-Kamal et al., 2005. Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. v 331, pp 428-434.

Slice et al., 2005. Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. v 280, pp 1582-1593.

Tang et al., 2005. The Adapter Protein CrkII Regulates Neuronal Wiskott-Aldrich Syndrome Protein, Actin Polymerization, and Tension Development during Contractile Stimulation of Smooth Muscle. J. Biol. Chem. v 280, pp 23380-23389.

Liu et al., 2004. Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase C&zeta, (PKC&zeta, ) and regulates PKC&zeta, activity. Mol. Cell. Biol. v 24, pp 6665-6675.

Sasai et al., 2004. The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. v 6, pp 741-748.

Tang and Gunst, 2004. The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. v 279, pp 51722-51728.

faqs

 

Question 1: I have high background and/or multiple bands on my western blot.  How can I fix this?

Answer 1:  There are multiple causes of high background and/or multiple bands.  Some suggestions to improve background signal include:

  1.   When blotting use 70v for 45min only as the small G-proteins are very mobile.
  2. Fully remove SDS from the gel by using a non-SDS containing buffer for transfer and performing a full 15 min gel wash step in the transfer buffer before blotting.
  3. Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
  4. Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
  5. Incubating with the primary antibody overnight at 4°, C and using the appropriate ECL detection system. 

 

Question 2: How much of the beads should I use for my pull-down experiments?

Answer 2:  PAK-PBD-GST beads (Cat. # PAK02) will bind to Cdc42-GDP with a much lower affinity than Cdc42-GTP.  If too many PAK-PBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) Cdc42.  The result of this will be an underestimation of Cdc42 activation.  For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Cdc42 activation or inactivation assay.  Once optimal conditions have been established, bead titrations should no longer be necessary.  We recommend 10, 15 and 20 &mu, g bead titrations.

 

Question 3:  How can I test whether the beads are working properly?

Answer 3:  A standard biological assay for PAK-PBD GST protein beads consists of a Cdc42 protein pull-down from cells loaded with either GTP&gamma, S (Cat. # BS01) or GDP.  Here are guidelines to follow (see Cat. # PAK02 or BK034 datasheets for more details):

 

Positive Cellular Protein Control:

Total cell lysate (300 &ndash, 800 &mu, g) should be loaded with GTP&gamma, S as a positive control for the pull-down assay.  The following reaction details how to load endogenous Cdc42 with the nonhydrolysable GTP analog (GTP&gamma, S).  This is an excellent substrate for PAK-PBD beads and should result in a strong positive signal in a pull-down assay.

 

a) Perform GTP loading on 300 &ndash, 800 &mu, g of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTP&gamma, S (200 &mu, M final concentration). Under these conditions, 5 - 10% of the Cdc42 protein will load with non-hydrolysable GTP&gamma, S and will be &ldquo, pulled-down&rdquo, with the PAK-PBD beads in the assay.

c) Incubate the control sample at 30°, C for 15 min with gentle rotation.

d) Stop the reaction by transferring the tube to 4°, C and adding 1/10th volume of STOP Buffer.

e) Use this sample immediately in a pull-down assay.

 

Negative Cellular Protein Control:

This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTP&gamma, S.  Loading endogenous Cdc42 with GDP will inactivate Cdc42 and this complex will bind very poorly to PAK-PBD beads.

 

 

If you have any questions concerning this product, please contact our Technical Service department at info@hoelzel.de

Hinweis: Die dargestellten Informationen und Dokumente (Bedienungsanleitung, Produktdatenblatt und Sicherheitsdatenblatt) entsprechen unserem letzten Update und sollten lediglich der Orientierung dienen. Wir übernehmen keine Garantie für die Aktualität. Für spezifische Anforderungen bitten wir Sie, uns eine Anfrage zu stellen.

Menge: KIT 50 assays
Lieferbar: In stock
Listenpreis: 860,64 €
Preis: 860,64 €
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