CD44, Abcam, ab254530

Our team has carried out deep research in the published scientific literature to identify papers and reports using antibodies directed against human CD44 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 32 commercial research antibodies against human CD44.

The collection of antibodies was tested using MDA-MB-231 WT and CD44 KO cells in the applications WB (WT and KO cell lysates/30 µg protein), IP (WT cell lysate), IF (WT and KO cells) and FC (WT and KO cells), furthermore, WB (WT and KO cell lysates/30 µg protein) for ab254530 was carried out using HAP1 WT and CD44 KO, according to YCharOS standard protocols (see reference).

In WB (1:5000 Ab dilution) ab254530 detected a single band of appr. 81 kDa in MDA-MB-231 WT lysate, with no corresponding band observed in MDA-MB-231 KO lysate. In the other WB (1:5000 Ab dilution) ab254530 detected a single band of appr. 81 kDa in HAP1 WT lysate with no corresponding band in KO lysate. A band was also detected following IP (2 µg Ab used). This band was more intense than the corresponding band in input extract (10% of starting material). Band was also detected in immunodepleted extract (10% of total unbound fraction), showing that the antibody does not completely deplete the antigen from the cell lysate. In IF (1:500 Ab dilution), a clear intense staining was observed in WT cells with no background staining in KO cells. Cell surface and intracellular flow cytometry (1 µg/mL antibody) showed a clear fluorescence shift in the WT cell population compared with the KO cells, indicating specific binding of the antibody to the target protein.

We highly encourage other scientists to share their observations.