This is a knockout-validated antibody summary, based on the publication "Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

[enlarge]

Figure 1. Protein immunoblot showing PLS1 is absent in hair bundles isolated from Pls1−/− mice. Molecular mass markers (in kD) are indicated. From [1].

Mouse monoclonal IgG2b

Company: Abnova

Antibody: PLS1

Catalog number: H00005357-M04

Summary: Mouse monoclonal IgG2b Kappa against partial recombinant PLS1 protein (a.a. 1-102). Reacts with human by western blot and ELISA.

[enlarge]

Figure 2. Labeling of utricle hair cells with Alexa Fluor 488–labeled anti-PLS1. Panels are 54 µm wide. (A) Wild-type littermate (Pls1+/+). (B) Heterozygote (Pls1+/−). (C) Homozygote null (Pls1−/−). From [1].

Western blot | Immunocytochemistry

Inner ear utricles and hair bundles from WT, Pls1-heterozygous and Pls1−/− mice. WB: mouse utricle hair bundles and whole utricles were mixed with NuPAGE 4× LDS sample buffer (NP0008; Thermo Fisher Scientific) and 10× reducing agent (NP0009; Thermo Fisher Scientific) were added to a final 1× concentration. IC: dissected utricles (with otoconia removed) were fixed for 20 min at room temperature in 4% formaldehyde (Electron Microscopy Sciences) in PBS.

WB: Prime Blocking Agent (RPN418; GE Healthcare).

IC: 5% normal donkey serum in PBS for 1 h.

IC: 1:250 dilution antibody conjugated to Alexa Fluor 488 dye using an Alexa Fluor 488 Antibody Labeling kit according to the kit’s instructions (Invitrogen) overnight at 4°C.

WB: Species-specific HRP-coupled secondary antibodies (115-035-003; Jackson ImmunoResearch Laboratories, Inc.).

WB: ECL Prime (RPN2232, GE Healthcare).

IC: at room temperature on a FluoView FV1000 laser scanning confocal microscope system (Olympus) and AF10-ASW 3.0 acquisition software, using a 60× 1.42 NA Plan-Apochromat objective with 4× zoom and 0.35-µm z-steps.

Antibody is also validated by IC, after conjugation to Alexa (to avoid secondary incubation).

The same clone (3G10) is sold as LifeSpan Biosciences LS-C197926; Abnova H00005357-M04; Sigma-Aldrich SAB1400214.