annexin A1 antibody | knockout validation | Invitrogen 71–3400
DOI
//dx.doi.org/10.13070/ko.en.6.1624
Date
2016-09-22

This is a knockout-validated antibody summary, based on the publication "Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

annexin A1 antibody | knockout validation | Invitrogen 71–3400 figure 1
Figure 1. Western blot showing B11, D6 and G5 KO clones for ANXA1, and compared with WT and PGS MIA PaCa-2, from figure 2 [1].
Antibody information

Company: Invitrogen

Antibody: ANXA1 (Annexin A1)

Catalog number: 71–3400

Summary: This product detects Annexin I in Human, Mouse and Rat samples. This product has been successfully used in ELISA, Flow Cytometry, Immunocytochemistry, Immunofluorescence, Immunohistochemistry (Paraffin) and Western Blot procedures. The immunogen is Full-length human recombinant Annexin I protein purified from yeast.

annexin A1 antibody | knockout validation | Invitrogen 71–3400 figure 2
Figure 2. Immunofluorescence analysis to detect ANXA1 in WT, PGS and ANXA1 KO MIA PaCa-2. Nuclei were stained with DAPI. From figure 2 [1].
Validation Method

WB and IC

Western Blot

Total intracellular proteins were extracted from the cells by freeze/thawing in lysis buffer containing protease inhibitors.

Protein content was estimated according to Biorad protein assay (BIO-RAD).

Samples (20 μg protein) were loaded onto 10% denaturing-polyacrylamide gel and separated by SDS-PAGE.

Incubation overnight at 4°C with the ANXA1 antibody (1:10000 dilution).

Membranes were blocked with 5% non-fat dry milk in TBS-Tween 20 (0.1% v/v).

Secondary incubation: RT with an appropriate secondary antibody (1:5000; Sigma-Aldrich).

Immunoreactive protein bands were detected by chemioluminescence using enhanced chemioluminescence reagents (ECL; Amersham). The blots were exposed to Las4000 (GE Healthcare Life Sciences).

Immunocytochemistry

WT, PGS and ANXA1 KO MIA PaCa-2 cells were fixed in p-formaldehyde (4% v/v in PBS; Lonza) and permeabilized with Triton X-100 (0.4% v/v in PBS; Lonza).

Blocked with goat serum (20% v/v in PBS; Lonza)

Incubated with anti-ANXA1 antibody (1:100 dilution).

Incubated with anti-rabbit AlexaFluor 488 (1:1000; Molecular Probes) for 2 hours at RT.

Samples were vertically scanned from the bottom of the coverslip with a total depth of 5 μm and a 63X (1.40 NA) Plan-Apochromat oil-immersion objective. Images and scale bars were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH) and presented as single stack.

References
  1. Belvedere R, Bizzarro V, Forte G, Dal Piaz F, Parente L, Petrella A. Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway. Sci Rep. 2016;6:29660 pubmed publisher