VAPB, Thermo Fisher Scientific, PA5-53023

Our team has carried out deep research in the published scientific literature to identify papers and reports using antibodies directed against human vesicle-associated membrane protein (VAMP)-associated protein B and C (VAPB) and followed up by extraction and evaluation of the available data to assess its performance according to pAbmAbs rating criteria.

YCharOS is here conducting a large head-to-head study comparing 6 commercial research antibodies against human VAPB.

The collection of antibodies was tested using HeLA WT and VAPB KO cells in the applications WB, IP and IF according to YCharOS standard protocols (see reference).

WB was performed using lysates (40 µg protein) from WT and KO cells. IP was performed using WT cell lysates (1 mg), and immunoprecipitated fraction was analysed by WB using anti-VAPB ab315013 (1:1000) from Abcam. IF staining was performed using WT and KO cells.

WB with PA5-53023 (antibody dilution 1:1500) produced a strong band between the 25 kDa and 35 kDa markers in lysates from HeLa WT cells, with no corresponding band observed in KO lysate. This matches the predicted size of 27.2 kDa for VAPB. In addition, several weak bands were observed in both WT and KO cells.

IP (2 µg antibody) yielded a distinct band at approximately 27 kDa, with higher intensity than the corresponding band in the input extract (4% of starting material). No band was observed in the unbound fraction (4% of total material), indicating complete immunodepletion of the antigen. In addition, a 25 kDa band was detected in the immunoprecipitated fraction, likely due to cross-reactivity of the WB secondary antibody with the Ig light chain.

In IF (1:250 antibody dilution), weak staining was observed in WT cells with no background staining in KO cells.

We highly encourage other scientists to share their observations.