This is a knockout-validated antibody summary, based on the publications "Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival" [1], and "The role of Rac1 in glycoprotein Ib-IX-mediated signal transduction and integrin activation" [2]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Company: BD

Antibody: Rac1

Catalog number: 610651

Mouse IgG2b,κ monoclonal antibody raised against human Rac1. Reacts with proteins of rat, human, mouse, dog, and chicken origin and has been successfully applied for western blot, immunofluorescence, and immunohistochemistry.

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Figure 1. Western blot of Rac1 in wild-type and Rac1 conditional knockout mice, from Figure 1 [1].

WB and IHC

6–8 intact postnatal day 1 (P1) lenses were pooled from both the WT and MLR-10/Rac1cKO mice.

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Figure 2. Immunohistochemical staining of Rac1 on paraffin sections from wild-type and Rac1 conditional knockout mice. Courtesy of the authors.

Pooled lens samples were homogenized in 10 mM Tris buffer pH 7.4 containing 0.2 mM MgCl2, 5 mM N-ethylmaleimide, 2.0 mM Na3 Vo4, 10 mM NaF, 60 μM phenyl methyl sulfonyl fluoride (PMSF), 0.4 mM iodoacetamide, Protease inhibitor cocktail tablets (complete, Mini, EDTA-free REF 11836170001) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (REF 04906837001) (1each/10 ml buffer, Roche (Basel, Switzerland)).

Homogenates were centrifuged at 800 × g for 10 min at 4 °C.

Protein concentration was estimated in supernatants using the Bio-Rad reagent (Cat. 500-0006).

This fraction was centrifuged further at 100,000 ×g for 1 h at 4 °C and the insoluble pellet derived was resuspended in the tissue homogenization buffer.

This centrifugation step was repeated twice and the resultant pellets were pooled and suspended in homogenization buffer containing 5 M urea,

enhanced chemiluminescence (ECL)

lens specific Rac1-deficient mice whole eyes (day 1).

Tissue sections derived from paraffin embedding were used in immunofluorescence analyses. Rac1 cKO and wild type (WT) littermate mice were fixed in 50 mM cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde, 4% sucrose and 2 mM CaCl2 for 2 h, and transferred to 10% buffered formalin.

paraffin sections at a 1:200 dilution for 24 h at 4 °C.

incubated in the dark for 2 h at room temperature (RT), with either Alexa fluor 488 or 594 conjugated secondary antibodies (Invitrogen).

Slides were mounted using Vecta mount and nail polish and photographed using a Nikon Eclipse 90i confocal laser scanning microscope.

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Figure 3. Western blot of Rac1 in platelets from wild-type and Rac1−/− mice, from Figure 1 [2].

WB, western blot

Rac1 KO mouse platelets were solubilized in SDS-buffer.

The article lists the antibody catalog number as 61051 instead of 610651.