Recombinant Anti-VCAM1 antibody [EPR5047] (ab134047)

ELISA using ab134047 at varying antibody concentrations and antigen concentration at 250 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

Lanes 1 - 6: Merged signal (red and green). Green - ab134047 observed at 105 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab134047 was shown to react with VCAM1 in treated wild-type A549 cells in Western blot with loss of signal observed in treated VCAM1 knockout cell line ab273758 (knockout cell lysate ab275504). Wild-type A549 and VCAM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab134047 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

ab134047 staining VCAM1 in HUVEC cells treated with TNF-α (ab55237) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134047 at 2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 647) (ab150119) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

Blocking/Diluting buffer: 5% NFDM/TBST.

Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples.

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

Blocking/Diluting buffer: 5% NFDM/TBST.

Immunohistochemical staining of paraffin embedded human tonsil with purified ab134047 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescent staining of K562 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134047 at a dilution of 1/250. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling VCAM1 with purified ab134047 at 1/40 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control. 

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

VCAM1 was immunoprecipitated from Human fetal liver lysate with ab134047 at 1/110 dilution.

Western blot was performed from the immunoprecipitate using ab134047 at 1/1000 dilution.

VeriBlot for IP secondary antibody (Peroxidase conjugated),was used as secondary antibody at 1/1000 dilution.

Lane 1: Human fetal liver lysate.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 1 second.

IHC image of VCAM1 staining in Human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

IHC image of VCAM1 staining in Mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.