Rab27A, Cell Signaling Technology, 95394

Our team performed a comprehensive review of the scientific literature to identify studies using antibodies against human Rab27A and Rab27B, followed by extraction and evaluation of available data according to pAbmAbs rating criteria.

YCharOS conducted a systematic head-to-head comparison of 14 commercially available antibodies against Rab27A and 7 against Rab27B across multiple applications

Antibodies were tested by WB using lysates (40 µg total protein) from U-87 MG WT cells and cells with siRNA-mediated knockdown (KD) of Rab27A or Rab27B. The same cell lines were used for IF testing. In addition, antibodies were evaluated by IP using HAP1 (Rab27A) or U-87 MG (Rab27B) cell lysates (1 mg), following YCharOS standard protocols (see reference).

In WB (1:200 antibody dilution), 95394 detects a single band at the expected size (~24 kDa) in WT U-87 MG lysate, with no corresponding band in lysates from Rab27A KD cells.

In addition, the antibody (1:200 dilution) detects a ~24 kDa Rab27A band in lysates (20 µg) from HAP1 WT, K-562, THP-1 (± PMA treatment), U-87 MG, HT-1080, U-2 OS, HeLa, and MDA-MB-231 cells. The intensity of the band is consistent with published Rab27A transcriptomic data (www.depmap.org) across these cell lines.

In IP, the antibody (2 µg) precipitates Rab27A, as detected by WB using the Rab27A-specific antibody 95394 from Cell Signaling Technology. However, the intensity of the precipitated band is comparable to that in the starting material and only slightly higher than in the unbound fraction, indicating partial but incomplete depletion of the target from the cell lysate.

In IF, the antibody (1:150 antibody dilution) resulted in a clear signal from U-87 MG WT and with no background signal in Rab27A KD cells.

We highly encourage other scientists to share their observations.