Glucagon receptor, Abcam, ab75240

We here review a publication from Novo Nordisk and University of Copenhagen providing a head-to-head comparison of all commercially available antibodies against the glucagon receptor (GCGR) (Bomholt et al., Communications Biology, 2022).
The authors systematically evaluated 12 commercial antibodies (Table S1) in HEK293 cells transfected with mouse or human cMyc-tagged GCGR cDNAs and studied liver sections from Gcgr+/+ and Gcgr-/- mice. Autoradiography, RNA-sequencing, and single cell RNA-sequencing were used as antibody independent approaches to support the findings obtained with IHC.
Transfected cells were permeabilized with methanol.
All GCGR antibodies, including this one from Abcam, gave overlapping staining with the cMyc antibody, demonstrating the ability to specifically recognize recombinant GCGR in transfected cells.
No staining was seen in control transfected cells or cells without primary antibodies (Fig. 1).
The antibodies were also checked for their ability to detect membrane bound GCGR by staining unpermeabilized HEK293 cells transfected with human GCGR and fixed in 4% PFA.
Abcam ab75240 (Antibody 11 in the paper) also showed positive staining of membrane bound GCGR (Fig. S1)
Abcam ab75240 was the only antibody in the study to show positive and specific staining of formalin-fixed and paraffin-embedded liver sections from female Gcgr+/+, not present in Gcgr-/- mice (Fig. 2a).
Ab75240 also showed some signal in IHC on paraffin embedded human liver tissue from patients with NASH (Fig. 2S).
Ab75240 was further tested in Western blotting using transfected HEK293 cells as samples. Mock transfected cells were used as negative controls. Here, ab75240 detected a band corresponding to the predicted size of GCGR (55 kDa) (Fig. 2b).
The specificity of ab75240 was further confirmed by Western blotting of liver tissues from Gcgr+/+ and Gcgr-/- mice, detecting a faint band of 55 kDa but also other bands of lower molecular weight, possibly due to proteolytic cleavage of GCGR. No bands were observed in Gcgr-/- tissue (Fig. S4).

In conclusion, Abcam ab75240 receives a 5 star rating in immunostaining based on its ability to specifically recognize GCGR in transfected cells, its positive signal in the human NASH samples, and positive staining of mouse WT liver, not present in KO liver. Ab75240 also receives a 5 star rating in Western blotting based on its ability to detect a band of the predicted molecular weight in samples from transfected cells and mouse liver, and the absence of the band in KO samples.
We highly encourage the scientific community to share their experiences.