GNB2 antibody | knockout validation | Abcam ab81272

This is a knockout-validated antibody summary, based on the publication "Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

GNB2 antibody | knockout validation | Abcam ab81272 figure 1
Figure 1. Immunoblots of indicated proteins in cells treated with DMSO or erlotinib (1 mM) for 6 hr to confirm specific knockout of negatively selected hits and on-target inhibition of EGFR pathway by erlotinib treatment. b-Actin was used as a loading control. Individual knockout cell lines were generated by lentivirus-mediated expression of sgRNA targeting indicated genes in HCC827 cells with constitutive Cas9 expression. From [1].
Antibody information

Rabbit monoclonal IgG

Company: Abcam

Antibody: GNB2

Catalog number: ab81272

Summary: Rabbit monoclonal IgG against a synthetic peptide within human GNB2 (aa 1-100, N terminal). Reacts with mouse, rat and human. Suitable for western blot, immunoprecipitation, immunohistochemistry (paraffin) and flow cytometry. Unsuitable for immunocytochemistry.

Validation Method

Western blot

Sample

Control and GNB2-KO HCC827 cells. Cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific #89901) supplemented with 100 x Protease Inhibitor Cocktail (Sigma #P8340), 100 x Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific #78427) and 25 units/ml Benzonase Nuclease (Sigma #E8263).

Blocking agent

5% Blotting-Grade Blocker (Bio-Rad #1706404) in Tris Buffered Saline with Tween 20, pH 8.0 (TBST) (Sigma #T9039) for 1 hr at room temperature.

Primary incubation

1:1,000 dilution in 5% BSA (Akron Biotechnology #AK8917-0100) overnight at 4°C.

Secondary incubation

Horseradish peroxidase (HRP) conjugated secondary antibody diluted in 5% Blotting-Grade Blocker.

Detection

Amersham ECL Western Blotting Detection Reagents (GE Healthcare #RPN2106) or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific #34580) and Amersham Hyperfilm ECL (GE Healthcare #28906839).

References
  1. Zeng H, Castillo Cabrera J, Manser M, Lu B, Yang Z, Strande V, et al. Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC. elife. 2019;8: pubmed publisher