osteocalcin antibody | knockout validation | Abcam ab13420
DOI
//dx.doi.org/10.13070/ko.en.7.2180
Date
2017-03-16

This is a knockout-validated antibody summary, based on the publication "Increased trabecular bone and improved biomechanics in an osteocalcin-null rat model created by CRISPR/Cas9 technology", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

osteocalcin antibody | knockout validation | Abcam ab13420 figure 1
Figure 1. Osteocalcin immunohistochemistry in WT and osteocalcin-null rats. Black arrows indicate positive (WT, left) and negative (null, right) osteoblasts. From [1].
Antibody information

Mouse monoclonal IgG3

Company: Abcam

Antibody: osteocalcin

Catalog number: ab13420

Summary: Mouse monoclonal IgG3 against full length cow Osteocalcin. Reacts with rat, sheep, rabbit, goat, chicken, cow, dog, human, and pig. Does not react with mouse. Suitable for western blot, immunohistochemistry (frozen and paraffin), immunocytochemistry/immunofluorescence and ELISA.

osteocalcin antibody | knockout validation | Abcam ab13420 figure 2
Figure 2. Western blot of femurs from compound heterozygous F2 animals demonstrate complete loss of osteocalcin protein. Beta-tubulin was used as loading control. From [1].
Validation Method

Western blot | Immunohistochemistry

Sample

Femurs from WT and osteocalcin-null rats. WB: Tissue samples were homogenized in ice-cold T-PER lysis buffer (Thermo Scientific, Waltham, MA) with a protease inhibitor tablet (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). IHC: femurs were fixed in 10% neutral buffered formalin (Sigma, St Louis, MO) and embedded in paraffin for sectioning.

Blocking agent

WB: 5% nonfat dry milk for 1 h at room temperature.

IHC: horse serum blocking solution (ImmPRESS system, Vector Laboratories) for 1 h.

Primary incubation

WB: 1:2,500 dilution at 4°C overnight.

IHC: 1:200 dilution in phosphate-buffered saline solution containing 1% bovine serum albumin.

Secondary incubation

WB: horseradish-peroxidase-conjugated goat anti-mouse-IgG secondary antibody.

IHC: appropriate secondary antibody (ImmPRESS, Vector Laboratories).

Detection

WB: chemiluminescence reagent (Thermo Scientific, Waltham, MA).

IHC: DAB (3,3′-diaminobenzidine) peroxidase substrate solution (Dako).

Clone note

The same clone (OCG3) is sold as Abnova MAB8377; Enzo Life Sciences ALX-804-536-C100; Abcam ab13420; Invitrogen 33-5700.

References
  1. Lambert L, Challa A, Niu A, Zhou L, Tucholski J, Johnson M, et al. Increased trabecular bone and improved biomechanics in an osteocalcin-null rat model created by CRISPR/Cas9 technology. Dis Model Mech. 2016;9:1169-1179 pubmed